In vivo active erythropoietin produced in insect cells

ABSTRACT

Disclosed and claimed is a human erythropoietin (EPO) expressed and produced in  Spodoptera frugiperda  Sf900+ cell line (ATCC: CRL 12579) transfected with a baculovirus construct containing the EPO gene. The EPO has an in vivo activity of 200,000 U/mg to 500,000 U/mg.

RELATED APPLICATIONS

Reference is made to, and this application claims priority from, U.S.application Ser. No. 60/162,354, filed Oct. 29, 1999 and U.S.application Ser. No. 60/118,816, filed Feb. 5, 1999, each of which ishereby incorporated herein by reference; and, each document cited inthose applications (“U.S. Ser. No. 60/118,816 appln cited document” and“U.S. Ser. No. 60/162,354 appln cited document”), and each documentcited or referenced in each U.S. Ser. No. 60/118,816 appln citeddocument and in each U.S. Ser. No. 60/162,354 appln cited document, ishereby incorporated herein by reference.

This application is also a continuation-in-part of U.S. application Ser.No. 09/169,178 filed Oct. 8, 1998 and issued as U.S. Pat. No. 6,103,526on Aug. 15, 2000.

Reference is made to U.S. application Ser. No. 08/965,698, filed Nov. 7,1997, Ser. No. 09/169,178, filed Oct. 8, 1998, Ser. No. 09/372,734,filed Aug. 11, 1999, Ser. No. 09/235,901, filed Jan. 22, 1999, Ser. No.09/169,027, filed Oct. 9, 1998, Ser. No. 08/120,601, filed Sep. 13, 1993(allowed), now U.S. Pat. No. 5,762,939, Ser. No. 08/453,848, filed May30, 1995 (allowed), now U.S. Pat. No. 5,858,368, Ser. No. 09/111,169,filed Jul. 7, 1998, and Ser. No. 08/430,971, filed Apr. 28, 1995(allowed; U.S. Pat. No. 5,976,552 issued Nov. 2, 1999), each of which ishereby incorporated herein by reference; and, each document cited ineach of these applications and patents or during their prosecution(e.g., as shown on the face of the patents or in their file histories)is hereby incorporated herein by reference. For instance, the presentinvention may be employed in practicing any or all of the aforementionedpatent applications or for otherwise expressing exogenous DNA or inproducing cells, e.g., for expressing exogenous DNA, for any or all ofthe aforementioned applications. Similarly, all documents cited in thistext (“herein cited documents”) and documents referenced or cited inherein cited documents or during the prosecution of any herein citeddocument (e.g., in the case of a herein cited document being a patent)are likewise incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods and apparatus for the growth ofcells, advantageously to high density, and uses thereof, including usesof the cells; and, products from the methods, apparatus and the cells.The present invention relates to methods and apparatus for the growth ofcells, advantageously of high density cells, for expression of exogenousDNA, such as from infection by a viral vector containing the exogenousDNA or by a plasmid transfected and/or inserted into such cells andcontaining such DNA, and uses thereof, including uses of the cells; and,products from the methods, apparatus and the cells and uses of suchproducts.

The present invention also relates to methods and apparatus for thegrowth and infection of insect cells, advantageously at high-density,and uses thereof, including uses of the cells; and, products from themethods, apparatus and the cells and uses thereof. Furthermore, theinvention relates to methods and apparatus for the production and use ofinsect cells, advantageously high density insect cells, for infectionwith wild type and/or genetically engineered recombinant baculoviruses,as well as to methods and apparatus for the production and use of cells,advantageously to high density, for infection, transfection or the likewith wild type and/or engineered recombinant vectors, e.g., viruses,plasmids, and uses thereof, including uses of the cells; and, productsfrom the methods, apparatus and the cells and uses thereof.

The methods and apparatus can include at least one bioreactor,advantageously at least one stirred-cell bioreactor, at least one sourceof culture medium (external to the bioreactor), advantageously at leastone source of stirred culture medium (external to the bioreactor), atleast one means for circulating media and/or cell culture, and at leastone means for dialysis of nutrients and waste (and/or extracellularexpression and/or secreted) products between the cells in the bioreactorand the external source of culture medium, such as at least onesemi-permeable membrane, e.g., a hollow fiber filter, that results inthe dialysis of nutrients and waste (and/or extracellular expressionand/or secreted) products between the cells in the bioreactor andculture medium; e.g., whereby there is a first loop between the culturemedium source and the dialysis means (media replenishment loop) and asecond loop between the bioreactor and the dialysis means (cell cultureloop).

The methods and apparatus can include at least one bioreactor,advantageously at least one stirred-cell bioreactor, at least one sourceof culture medium (external to the bioreactor), advantageously at leastone stirred source of culture medium (external to the bioreactor), atleast one means for circulating media and/or cell culture, and at leastone means for delivery of oxygen, such as at least one oxygenatorincluding input and output ports.

The methods and apparatus can include at least one bioreactor,advantageously at least one stirred-cell bioreactor, at least one sourceof culture medium (external to the bioreactor), advantageously at leastone source of stirred culture medum (external to the bioreactor), atleast one means for circulating media and/or cell culture, at least onemeans for dialysis of nutrients and waste (and/or extracellularexpression and/or secreted) products between the cells in the bioreactorand the external source of culture medium, such as at least onesemi-permeable membrane, e.g., a hollow fiber filter, that results inthe dialysis of nutrients and waste (and/or extracellular expressionand/or secreted) products between the cells in the bioreactor andculture medium, e.g., whereby there is a first loop between the culturemedium source and the dialysis means (media replenishment loop) and asecond loop between the bioreactor and the dialysis means (cell cultureloop), and optionally but advantageously present, means for delivery ofoxygen; for instance, via means comprising at least one oxygenatorincluding input and output ports. Advantageously, oxygen is delivered ina way such that proper oxygenation of the cells is maintained at celldensities especially at high densities. A “source of culture medium” or“culture medium source” can be a vessel for culture medim. A bioreactorcan be a vessel for cells or cell culture.

Further, the methods and apparatus can include means for the delivery ofother gases, such as air, and/or nitrogen, and/or carbon dioxide. Themethods and apparatus also can include means for monitoring of chemicaland/or physical parameters, such as pH and/or conductivity and/ortemperature and/or oxygen concentration and/or carbon dioxideconcentration and/or nitrogen concentration and/or glucose/nutrientconcentration. And, the methods and apparatus can include means foradjusting one or more chemical and/or physical parameters of the systemsuch as a function of one or more monitored parameters, e.g., pH and/ortemperature and/or oxygen concentration and/or carbon dioxideconcentration.

The methods and apparatus can optionally include means for monitoringand/or probing the system such as probe port(s); and further optionallymeans for delivery of at least one additional gas such as air and/ornitrogen and/or carbon dioxide; and still further optionally means formonitoring and/or regulating other parameters such as pH and/ortemperature.

Accordingly, the invention can relate to a method for growing cellscomprising culturing cells in at least one bioreactor whereby there is acell culture, supplying medium in at least one vessel whereby there isculture medium, circulating culture medium and/or cell culture, wherebythe bioreactor and vessel are in fluid communication and the cellculture and/or culture medium are in circulation, and delivering oxygento the cell culture and/or culture medium.

And, the invention also can relate to a method for growing cellscomprising culturing cells in a bioreactor whereby there is a cellculture, supplying culture medium in a vessel where by there is culturemedium, circulating the cell culture through a dialysis means,circulating culture medium through the dialysis means, wherein thedialysis means in fluid communication with the bioreactor and thevessel, whereby there is a first, cell culture, loop between thebioreactor and the dialysis means, and a second, media replenishment,loop between the vessel and the bioreactor, and the method includesperforming dialysis between the culture medium and the cell culture.

Other aspects of the invention are described in or are obvious from (andwithin the ambit of the invention) the following disclosure.

BACKGROUND OF THE INVENTION

Biological substances derived from animal cell cultivation are findinguses in a variety of medical and agricultural applications. Theimportance of recombinant proteins, a specific subset of biologicalsubstances, has been the basis for many new and emerging therapies anddiagnostic methodologies ranging from vaccines to cancer therapies.

Cell culturing processes for the production of biological substancesrange in complexity from simple manually operated batch processes tocomplex computer controlled continuous cultivation bioreactors; forinstance, from simple 50 mL spinner flasks to complex stirred-tankbioreactors of 500 L or more with automatically operated multiplemeasurement devices and feedback controls. The basic principle behindeach process is to utilize cells as catalytic engines to produce usefulbiological substances such as viruses or proteins using medium in whichthe cells are bathed to provide both a source of required nutrients anda means of removing inhibitory waste material.

As the production of biological substances moves from the researchlaboratory to commercial production, competitive markets demandproductivity improvements. The yield of product from each commercialbioreactor becomes critical. So to with quality, the market demandsreliability and consistency of output. Current cell culturing processesreadily reach their limiting conditions for production of biologicalsubstances. These limitations are imposed by the nutrient and oxygenrequirements of the cells and by accumulation of inhibitory wastemetabolites; and are reached well before the theoretical limits of cellgrowth or protein production are reached.

Not all cell types are capable of producing all biological substances.Many biological substances found in certain cells are incompatible withor even toxic to other cell types. The choice of cell types in manysituations depends on the structural complexity of the end protein beingproduced. While protein production levels are high in prokaryoticorganisms given their rapid growth and concomitant high levels ofprotein expression, they are not always capable of producing functionalproteins as they perform no or incomplete or differentpost-translational and/or co-translational modifications such asglycosylation, phosphorylation and complex multi-unit macro-assembly.

Animal cells do perform the necessary complex post-translationalmodifications including glycosylation, phosphorylation andmacro-assembly. However, some animal cells, especially mammalian cells,are difficult to grow and maintain and do not readily lend themselves tohigh yield production of biological substances under industrialconditions. As a subset of animal cells, insect cells are capable ofglycosylation, phosphorylation and macromolecular assembly. For theproduction of many recombinant proteins, insect cells are an excellentchoice because these cells have simple growth requirements, are highlysusceptible to infection by recombinant baculoviruses engineered toproduce biological substances in insect cells, and have a good safetyprofile.

Cell types and desired growth dynamics dictate the selection of abioreactor type. Basic bioreactor devices include culture flasks, rollerbottles, shaker flasks, stirred-tank reactors, air-lift reactors andmore recently, hollow fiber reactor devices. There are advantages anddisadvantages to each type of bioreactor and these advantages anddisadvantages vary according to the type of cell cultured in the systemand the specific properties of those cells. What works well withattached cells may not with suspended cells. Therefore, improvedbioreactors need to be flexible. They should support various cell types,operate for short or long duration cultivation periods and shouldoperate at scales ranging up to 10,000 liters.

Growth of attached cells is limited to the surface area available andwhen roller bottles are used, scale up of attached cell production ofbiological substances can demand significant amounts of space.Alternatively, for attached cells, microcarriers can be used. However,these can limit nutrient and oxygen availability to the cells and oftenexpose them to additional sheer forces as the use of microcarriersrequires a stirred tank. Additionally, matching the proper microcarriertype to the specific cell type can prove difficult.

Insect cells represent an economically important cell type withdemonstrated usefulness in manufacturing biological substances.Typically, insect cells are cultured as suspensions in stirred cellbioreactors.

Unlike bacteria that are enclosed in cell walls, animal cells, andspecifically insect cells, respond negatively to relatively mildhydrodynamic shear forces found in an operating bioreactor. Thesedamaging events include bulk-fluid turbulence associated with spinnervortex formation, fluid-tank wall collisions and gas/liquid interfaces.This gas/liquid interfaces include the interface between the culturemedium and head space gas with the stirred tank and between culturemedium and oxygen bubbles formed during oxygen addition, such as withsparging. Insect cells are more sensitive than many other animal cellsto these hydrodynamic shear forces (Wu J, King G, Daugulis A. J.,Faulkner O, Bone D. H., Goosen M. F. A. (1989) Applied Microbiology andBiotechnology 32: 249). Compounding this sensitivity is the requirementof insect cells for higher oxygen levels: introduction of oxygenproduces more bubbles, that is, more gas/liquid interface, and theopportunity for more hydrodynamic shear damage.

Thus, with insect cells, the mechanism for adding oxygen to the systembecomes critical. First, the cells are more sensitive to the shearforces than are other animal cells. Second, more oxygen is required togrow these cells than is required to grow other animal cells. Thisadditional oxygen requirement brings with it the probability of furthercell destruction associated with increased bubbling from the higheroxygen supply and with faster stirring required to ensure even oxygendistribution. And third, when infected with baculovirus, the oxygendemand increases yet again and so too, the probability for shear relateddamage increases with a third factor.

Cell death is the end result of excessive shear forces, resulting fromloss of membrane integrity, cell lysis, and altered metabolic activity.This insect cell sensitivity to shear forces related to high oxygenrequirement is evidenced by the need for surfactant addition to theculture medium in sparged stirred tank bioreactors of any size(Murhammer D. W., Goochee C. F. (1990) Biotechnology Progress 6: 391).

During the cell culturing processes, oxygen demand increases as celldensity increases. If the oxygen need is met through increased oxygenflow and stirring, shear forces increase. Thus, oxygen remains one theof key limiting factors in high density cell culture due to the need tolimit shear related cell death. In turn, limiting oxygen additionrestrains cell growth and makes high density culture unattainable.Furthermore, poor oxygenation directly limits output of recombinantprotein with insect cell based cell culturing systems.

Thus, it would be an advance in the art to address issues that limitcell density and recombinant protein production, such as providing botha source of required nutrients and a means of removing inhibitory wastematerial and/or providing oxygenation that addresses the desire toreduce or limit shear related cell death from oxygenation.

Zhang et al. Biotech. Bioeng. 59(3): 351–9 (1998) relates to ahigh-density insect cell perfusion process utilizing an ultrasonicfilter device as a means to retain cells within the bioreactor whileextracting spent medium. Per cell yields of recombinant protein weresimilar between normal conditions (when cells were diluted to a lowdensity and infected with a genetically engineered baculovirus) andhigh-density conditions, and thus failing to demonstrate, show, teach orsuggest production of a recombinant protein at high cell density. And,in a perfusion system, nutrients and waste never approach equilibrium.Thus, Zhang et al. either individually or in any combination fails toteach or suggest the present invention.

Likewise, any other filters or hollow fibers or hollow fiber filterdevices or uses thereof fail to teach or suggest the present invention.For instance, in contrast with certain embodiments of the presentinvention, filters or hollow fibers or hollow fiber filter devices canbe used: by removing medium and the cells from the bioreactor vessel,passing it through the filtering device, collecting the perfused fluidcontaining the desired biological substance and returning the mediumwith its cells to the original bioreactor vessel; or as housing forcells of interest within the extra-lumenal space of a hollow fiberfilter device with perfused medium passed through the capillary tubes tothe cells; or by placing unencased hollow fibers directly into thefermentation tank itself so that fresh medium can be more directlyprovided to immobilized or attached cells.

Microbead encapsulation involves porous hollow microballoons. Culturecells attach to the internal surfaces of these porous hollowmicroballoons. By controlling the diameter of the microballoon and itspore sizes, relative to cell size, the thickness of the cell layers canbe controlled to allow for adequate delivery of nutrients and removal ofwaste metabolites. Microbead encapsulation fails to teach or suggest thepresent invention.

Spaulding et al., U.S. Pat. No. 5,637,477, concerns a process for insectcell culture that reduces shear, in a horizontally rotating culturevessel. Spaulding et al. too, either individually or in any combinationfails to teach or suggest the present invention.

Goffe, U.S. Pat. No. 5,882,918 relates to a cell culture incubator.There is no circulation of cells. Goffe, either individually or in anycombination, fails to teach or suggest the present invention.

Portner et al. Appl Micro Biot. 403–414 (1998) is directed to dialysiscultures and involves a complicated dialysis process coupled with theperfusion of waste and the addition of nutrient concentrate(s) as ameans to reach high cell densities wherein the removal of waste is donein a dialysis vessel connected to a semi-permeable membrane and twoadditional vessels (one for the addition of dialyzing fluid and thesecond for the removal of waste). As a result, some nutrients must alsodialyze into the dialysis vessel and get wasted. Further, one or moreconcentrates are added directly to the culture vessel to add nutrientsand support the growth of cells and to replace what is being lost in thedialysis compartment of the bioreactor.

Portner et al. state that a limitation of their design when used in astirred tank bioreactor is oxygen limitation in their dialysis loop (p.409). Further, in one example with mammalian cells (p. 410, hybridomacells), Portner et al. give no data or any indication that cellsactually grew to high density; and in fact, the yields of monoclonalantibodies they report after 850 hours of culture (35.4 days) wererelatively low (478 mg/l or 13.8 mg/l/day). Further, Portner state intheir conclusions (p. 412) that their dialysis bioreactor can be usedwith stationary animal cells and that for large-scale cultures ofsuspended cells, that an external loop can “lead to severe problems,mainly due to oxygen limitations in the loop.”

Thus, Portner et al. directly teach away from the present invention bydirectly teaching that a bioreactor with an external loop of circulatingcells will not work. Moreover, Portner relates to the use of an openbioreactor system requiring constant addition of dialyzing fluid to adialysis chamber and nutrient concentrates to the bioreactor. Continuousperfusion of the dialysis chamber is a variation on a perfusion systemin which nutrients and waste never approach equilibrium. And, Portner etal. do not teach or suggest the addition of oxygen by in line spargingor other means, suggesting that external circulation of cells is limitedby oxygen depravation.

Garnier et al., Cytotechnology 22: 53–63 (1996) relates to dissolvedcarbon dioxide accumulation in a large scale and high density productionof TGFβ receptor with baculovirus infected Sf-9 cells: Aerationapparently involved accumulation of dissolved carbon dioxide thatinhibited protein production; oxygen may serve as a carrier gas fordesorbing carbon dioxide. Garnier used a low flow rate of pure oxygenwith a dissolved oxygen content of 40%, and shows that there was aproblem in the art, namely that higher rates of oxygen addition canresult in hydrodynamic stress detrimental to the culture. Garnier failsto teach or suggest how one could provide higher rates of oxygentransfer, or to balance oxygen transfer, mechanical stress and carbondioxide, inter alia. Garnier fails to teach or suggest the addition ofoxygen by in line sparging or other means of the present invention, aswell as the apparatus and methods of the present invention, inter-alia.

Karmeu et al. Biotechnology and Bioengineering 50: 36–48 (1996) isdirected in on-line monitoring of respiration in recombinant-baculovirusinfected and uninfected insect cell bioreactor cultures. Dissolvedoxygen (DO) levels were generally at about 40%, and as to DO, theauthors assert that further investigations are required to clarify theeffect of DO on baculovirus-infected insect cells. Karmeu et al. mayprovide that respiration in insect cell cultures can be continuouslymonitored on-line with data from an O₂ control system or an IR CO₂detector; but, fails to teach or suggest the system and apparatus of thepresent invention, especially the addition of oxygen by in line spargingor other means of the present invention (alone or in combination withdialyzing means), dialyzing means (alone or in combination with oxygenaddition means) as in the present invention as well as other apparatusand methods of the present invention, for instance, use or adjusting ofCO₂ in response to pH changes inter alia (and indeed, Karmeu teachesaway from such by reporting that insect cell cultures reportedly do notrequire HCO₃ ⁻/CO₂ buffering).

Nakano et al. Appl Microbiol Biotechnol 48(5): 597–601 (1997) relates tothe influence of acetic acid on the growth of E. coli during high-celldensity cultivation in a dialysis reactor with controlled levels ofdissolved oxygen with different carbon sources (glucose and glycerol);but fails to teach or suggest methods and apparatus of the invention.

Gehin et al. Lett Appl Microbiol 23(4): 208–12 (1996) concerns studiesof Clostridium cellulolyticum ATCC 35319 under dialysis and co-cultureconditions. This was in batch with and without pH regulation. H₂, CO₂acetate, ethanol and lactate were end-products. No synergistic actionwas found. Methods and apparatus of the invention are not taught orsuggested by Gehin.

Schumpp et al. J Cell Sci 97(Pt4): 639–47 (1990) relates to cultureconditions for high cell density proliferation of HL-60 humanpromyelocytic leukemia cells. While nutrient supply and metabolic endproduct accumulation are possible growth limiting factors, Schumppfavors a perfusion method. Accordingly, methods and appartus of theinvention are not taught or suggested by Schumpp.

LaIuppa et al., “Ex vivo expansion of hematopoietic stem and progenitorcells for transplantation,” in Jane N. Winter (ed.), Blood Stem CellTransplantation, 1997 illustrates various systems for expansion ofhematopoietic stem and progenitor cells, and fails to teach or suggestmethods and apparatus of the invention.

Bedard et al., Biotechnology Letters, 19(7): 629–632 (July 1997)concerns fed batch culture of Sf-9 cells which reportedly supported3×10⁷ cells per ml and improved baculovirus-expressed recombinantprotein yields; and relates to Sf-900 II medium and nutrient additivesand nutrient concentrates. While medium, additives and nutrientconcentrates may be employed in the practice of the herein invention,Badard et al. fails to teach or suggest methods and apparatus of theinvention. Indeed, more generally, while components and/or cells foundin literature, such as herein cited literature, may be employed in theherein invention, it is believed that heretofore methods and appartus ofthe invention have not been taught or suggested.

Accordingly, it is believed that heretofore simple systems, e.g. closedsystems, as in the present invention, where, for instance, nutrients andwaste products in the bioreactor and the dialysate are in equilibriumand do not necessitate continuous perfusion (dialysis used not only forremoval of waste but for addition of nutrients) and/or the issue ofoxygen depletion is addressed, e.g., by the addition of oxygen directlyto circulating cells, with also the issue of reducing or limiting shearrelated cell death due to oxygenation by reducing or limiting oreliminating shear forces from oxygen addition addressed, have not beentaught or suggested. And, it is believed that heretofore, new bioreactorsystems and apparatus for high-density cell growth, uses thereof,products therefrom, as described and claimed herein, as well as theherein methods for making and using such a high-density cells andproducts therefrom, have not been disclosed or suggested in the art.

OBJECTS AND SUMMARY OF THE INVENTION

An object of the invention can be to provide an apparatus and/or aprocess for the growth of cells and/or of cell products, for instance,to high density.

The apparatus and process can include the use of a dialysis procedurefor the simultaneous removal of waste products and the replacement ofnutrients during the growth of cultured cells. The dialysis procedurecan employ the circulation of the growing cells through a semi-permeablemembrane, such as a hollow fiber filter, where there is the exchange ofsmall molecules between the cell medium and an external source ofaddition medium, referred to as ‘regeneration’ medium or media.Semi-permeable membranes permit the passage of water and small moleculesand smaller proteins but not cells. If the concentration of a smallmolecule increases or decreases on either side of the membrane, then theconcentration gradient leads to the exchange of molecules across thesemi-permeable membrane. This provides for removal of waste moleculesout of the cell compartment along a concentration gradient and entry ofreplacement nutrients into the cell compartment along a differentconcentration gradient. Where the membrane is essentially inert, as in ahollow fiber filter, then the movement is driven by the diffusion ofmolecules across the membrane and requires no specific pressurization todrive the molecules across the membrane.

The apparatus and process can provide a modular set of interchangeablecomponents. This interchangeability can provide for optimization duringdifferent phases of a cell cultivation run to improve performance andfor the capability for rapidly exchanging a malfunctioning componentwithout aborting a cell cultivation run.

The circulation of cells through the relatively small diameter tubing ofthe hollow fiber filter provides the additional advantage of disruptingany clumped cells. Clumped cells are not as efficient in producingproduct since the interior cells of a clump cannot as easily absorbnutrients and oxygen and eliminate waste products as the outer cells.

Another object of the invention can be to provide an apparatus and aprocess for the addition of oxygen to growing cells using a novelprocedure where the addition of oxygen is done outside the bioreactorand into a circulating loop of cells. This process is referred to as‘in-line’ oxygenation. A means of introducing the oxygen gas is tocirculate the cells through a hollow fiber filter designed for theaddition of oxygen to fluid, such as the UniSyn Technologies Oxy 1.Alternatively and/or additionally, oxygenation can be accomplished bydirect sparging of the circulating loop of cells and/or with isolatedfibers within the hollow fiber filter device used in medium exchangededicated to oxygen exchange and/or through sparging of the“replenishment” medium and/or through at least one oxygen-containingcompound that releases dissolved oxygen and/or any combination of theseoxygenation means.

An embodiment of the invention is the use of insect cells in a processthat provides for their growth to high density; however, the inventionis applicable to any cells, e.g., typical cells used in expressionsystems (see infra).

A further object of the invention can be to use cells, such as insectcells or cells used in expression systems at high density with any, orall, and advantageously most or all, of the following characteristics:replicate continuously in suspension as single cells, making them idealfor use in large-scale pharmaceutical bioreactors; grow to high densitywith a high degree of viability in a low-cost, serum-free medium;support the replication of vectors, e.g., baculoviruses, to high titers;when infected with a genetically engineered recombinant vector, e.g.,baculovirus, gene; produce products at high levels and produce thoseproducts consistently over many passages; meet all regulatoryrequirements for identity and safety; readily expand to large-scalebioreactors for the manufacture of pharmaceutical products; and, storeand culture in a serum-free medium.

Yet another object of the invention can be to provide a bioreactor and aprocess which overcomes or addresses at least one or more problem(s) ofprior bioreactors and processes, e.g., problems identified herein withprior high-density bioreactor processes.

Surprisingly it has been found that the herein apparatus and processwill grow cells such as insect cells or cells used in expression systemsto high density and make them ideal for use in the large-scaleproduction of gene products for use in human and animal health. At highcell density, the cells grow continuously as single cell suspensions ina commercial serum-free medium, divide rapidly and maintain a high levelof viability, and are highly permissive for infection or transfectionwith vectors, e.g., baculoviruses, producing high virus titers and highlevels of recombinant gene products. In addition, the herein bioreactorand process can be used with Sf900+ insect cells that meet therequirements for identity and safety recommended for the manufacture ofrecombinant DNA gene products under the U.S. current Good ManufacturingPractices (cGMP) specifications (Code of Federal Regulations 21, Part211, Current Good Manufacturing Practice for Finished Pharmaceuticals,Apr. 1, 1995). The Sf900+ cells are also in compliance with theguidelines issued by the U.S. Food and Drug Administration Points toConsider for Cell Lines used in the Production of PharmaceuticalProducts (Points to Consider in the Characterization of Cell Lines Usedto Produce Biologicals, issued May 17, 1993, U.S. Food and DrugAdministration, Rockville, Md.).

Thus, an embodiment of this invention can be a process for the growth ofcells, e.g. the insect cells Sf900+, to high cell densities.

Another object of the invention can be to provide different media duringthe course of cell culture. The purpose is to change medium compositionduring different phases of cell culture to optimize nutrientutilization. For example, a “growth” media would be optimized for growthof cells to high density while an “expression” media would be optimizedfor the expression of biological substances in the cells. It can be afurther object of the invention that the “expression” media be a lowcost formulation composed of carbohydrates and organic and inorganicsalts. This media thus reduces the cost of production of a biologicalsubstance. Additionally, since more complex media often containsubstances that are difficult to separate from the desired product,simple “expression” medium allows for easier purification, reducing costyet again.

Another embodiment of this invention can be to provide a method to usethe high-density cells for the production of high titers of wild typeand genetically engineered recombinant vectors, e.g., baculoviruses.

Yet another embodiment of this invention can be to provide the use ofthe bioreactor and process to produce high density cells to makevectors, e.g., expression vectors, such as baculovirus expressionvectors, and to produce high-titer stocks of recombinant virus or vectorsuitable for use in the production of recombinant gene products.

Still another embodiment of this invention can be to provide thebioreactor and process to produce cell lines conforming to standardtests for identity and safety, whereby the cells can be used in thecommercial manufacture of pharmaceutical products.

And, another embodiment of this invention can be to provide a bioreactorand method for the production of cells such as insect cells forlarge-scale commercial production of recombinant gene products fromexpression vectors such as baculovirus expression vectors.

The inventive bioreactor and process for high cell density is especiallysuited for practicing the teachings of the applications and patentsabove-referenced under “Related Applications”; and, this provides yetfurther embodiments of the invention.

Accordingly, in certain aspects, the invention can entail apparatus andprocess for producing high densities of cells. The invention, in certainaspects, can also comprehend the use of a high density process for thegrowth of an insect cell line such as an insect cell line establishedfrom Lepidoptera, Noctuidae, Spodoptera frugiperda Sf900+ (ATCC: CRL12579) in a serum-free insect medium supplemented. The invention, incertain aspects, can also comprehend an expression system such as abaculovirus expression system, including a recombinant virus or vector,e.g., baculovirus, that includes exogenous coding DNA, wherein cellssuch as insect cells, at high density from inventive apparatus andmethods are infected or transfected with the recombinant vector orvirus, e.g., baculovirus.

Further, the invention provides an apparatus for growing cellscomprising at least one bioreactor for cell culture, at least one vesselfor culture medium, means for circulating culture medium and/or cellculture, whereby the bioreactor and vessel are in fluid communication,and at least one means for delivery of oxygen. The invention furtherprovides an apparatus comprising a bioreactor for cell culture, a vesselfor culture medium, means for circulating cell culture, means forcirculating culture medium, dialysis means in fluid communication withthe bioreactor and the vessel, whereby there is a first, cell culture,loop between the bioreactor and the dialysis means, and a second, mediareplenishment, loop between the vessel and the bioreactor, and inoperation dialysis between the culture medium and the cell culture; and,this apparatus can further comprise at least one means for delivery ofoxygen into the cell culture loop.

The means for delivery of oxygen comprises a hollow fiber filteroxygenator and/or means for delivery of oxygen comprises means forin-line sparging and/or means for delivery of oxygen comprising meansfor delivery of at least one oxygen-containing compound that releasesdissolved oxygen into cell culture. The means for delivery of oxygen canbe positioned upstream of input of circulating cell culture returning tothe bioreactor. The bioreactor and/or the vessel; and advantageouslyboth the bioreator and the vessel, are stirred. The means for deliveryof oxygen can provide an average dissolved oxygen concentration of about60% and/or greater than 60% or 65%; and/or the means for delivery ofoxygen can provide an average dissolved oxygen concentration of greaterthan about 40% and/or the means for deliver of oxygen can provide anaverage dissolved oxygen concentration between about 30% and 90% orbetween about 40% and about 80% or between about 50% and 70%.

The apparatus can further comprise means for measuring physical and/orchemical parameters of the cell culture and/or the culture medium; forinstance, in the cell culture loop and/or the media replenishment loop,such as probes or sensors in the bioreactor or the vessel or at anysuitable point in the loop(s) (for instance, where there is withdrawalfrom the loops such as for sampling). The means for measuring cancomprise means for measuring dissolved oxygen concentration; e.g., inthe cell culture or cell culture loop, for instance, a probe or sensorin the bioreactor for detecting dissolved oxygen in the cell culturetherein. The means for measuring can comprise means for measuring pH;e.g., in the cell culture or cell culture loop, for instance, a probe orsensor in the bioreactor for detecting pH. The means for measuring cancomprise means for measuring temperature; e.g., in the cell culture orcell culture loop, for instance, a probe or sensor in the bioreactor fordetecting temperature. The means for measuring can comprise means formeasuring pH and means for measuring dissolved oxygen; e.g., in the cellculture or cell culture loop, for instance, probes or sensors in thebioreactor for detecting each of pH and dissolved oxygen. The means formeasuring can comprise means for measuring and/or counting cell densityor cells.

The apparatus can further comprise means for adjusting physical and/orchemical parameters of the cell culture and/or the culture medium inresponse to data from the measuring means. The adjusting means cancomprises means to adjust temperature, such as a heating and/or coolingjacket in surrounding relationship with the vessel and/or the biorectorconnected to a computer, microprocessor or processor that provides asignal to the jacket for heating and/or cooling in response totemperature measurements varying from a desired level. The adjustingmeans comprises means for adjusting pH; such as means for adding achemical to the cell culture and/or the media that alters pH thereinconnected to a computer, microprocessor or processor that provides asignal to the adjusting means for addition of the chemical in responseto pH measurements varying from a desired level, for instance, means foradding carbon dioxide to the cell culture in response to pHmeasurements. Thus, the adjusting means also can comprise means foradjusting dissolved carbon dioxide concentration. Further, the adjustingmeans can comprise means for adjusting dissolved oxygen concentration;for instance, means for addition of oxygen and/or air (or both) inresponse to oxygen measurements varying from a desired level (such as alevel between 30% and 90% such as between 40% and 80% for instancebetween 50% and 70%, e.g., approximately 60%). In addition and/oralternatively, the adjusting means can call for adjusting and/orchanging conditions in response to a cell density and/or cell countmeasurement; for instance, at a particular cell and/or cell count, mediamay be changed and/or a vector (e.g., recombinant virus such asbaculovirus) added for infection.

Advantageously, the adjusting means comprises means for adjustingdissolved oxygen and means for adjusting dissolved carbon dioxide,whereby in response to pH measurement(s), dissolved carbon dioxidelevels are adjusted; and, even more advantageously, the adjusting meansalso includesmeans for adjusting dissolved oxygen in response todissolved oxygen measurement(s). These “adjustments” are advantageouslyperformed in the cell culture loop; e.g., addition of carbon dioxide andoxygen are performed in the cell culture loop, for instance, at theoxygenator. The pH can be set to a desired level and carbon dioxideadjusted when pH varies from the desired level, whereby the dissolvedoxygen measurement varies periodically as a function of time. Forinstance, the dissolved oxygen measurement varies from 30% to 90% orfrom 40% to 80% or from 50% to 70%; or, the dissolved oxygen measurementaverages about 60% and/or the dissolved oxygen measurement can vary fromhigh value to low value over about 10 to about 30 minutes or over about20 minutes and/or a plot of the dissolved oxygen measurement as afunction of time comprises a sin wave.

The invention yet further comprehends methods involving the inventiveapparatus or steps performed by the apparatus or analogous apparatus.

The invention still further provides a method for growing cellscomprising culturing cells in at least one bioreactor whereby there is acell culture, supplying medium in at least one vessel whereby there isculture medium, circulating culture medium and/or cell culture, wherebythe bioreactor and vessel are in fluid communication and the cellculture and/or culture medium are in circulation, and delivering oxygento the cell culture and/or culture medium. The invention also provides amethod for growing cells comprising culturing cells in a bioreactorwhereby there is a cell culture, supplying culture medium in a vesselwhere by there is culture medium, circulating the cell culture through adialysis means, circulating culture medium through the dialysis means,wherein the dialysis means in fluid communication with the bioreactorand the vessel, whereby there is a first, cell culture, loop between thebioreactor and the dialysis means, and a second, media replenishment,loop between the vessel and the bioreactor, and the method includesperforming dialysis between the culture medium and the cell culture.

The delivering of oxygen can be by means for delivery of oxygencomprising a hollow fiber filter oxygenator and/or by means for in-linesparging and/or for delivery of at least one oxygen-containing compoundthat releases dissolved oxygen into cell culture; and, oxygen can bedelivered into the cell culture and/or the cell medium; advantageouslyinto the cell culture; for instance, into the cell culture loop, such asimmediately prior to return of cell culture to the bioreactor, e.g.,upstream of input of circulating cell culture returning to thebioreactor.

The method can further comprise stirring the cell culture or the culturemedium or, advantageously, both the cell culture and the culture medium.

The delivering of oxygen can provide an average dissolved oxygenconcentration of about 60% or greater than about 60% or greater thanabout 65%; and/or an average dissolved oxygen concentration of greaterthan about 40%; and/or the delivering of oxygen can provide an averagedissolved oxygen concentration between about 30% and 90% or betweenabout 40% and about 80% or between about 50% and 70%.

The dialysis means can comprise at least one semi-permeable membrane.The semi-permeable membrane can comprise at least one hollow fiberfilter.

Furthermore, the methods can include delivering oxygen into the cellculture loop; for instance, the delivering of oxygen can be by means fordelivery of oxygen comprising a hollow fiber filter oxygenator and/or bymeans for delivery of oxygen comprising means for in-line spargingand/or the delivering of oxygen can comprise delivering at least oneoxygen-containing compound that releases dissolved oxygen into cellculture. The delivering of oxygen can by means for delivery of oxygen ispositioned upstream of input of circulating cell culture returning tothe bioreactor.

Further still, the methods can include measuring physical and/orchemical parameter(s) of the cell culture and/or the culture medium. Themeasuring can comprise measuring dissolved oxygen concentration and/ormeasuring pH and/or measuring temperature; and/or measuring pH andmeasuring dissolved oxygen concentration and/or measuring cell densityand/or amount of cells.

Even further still, the methods can include adjusting physical and/orchemical parameters of the cell culture and/or the culture medium(advantageously the cell culture) in response to data from themeasuring; for instance, the methods can include adjusting temperatureto maintain a desired temperature and/or adjusting pH to maintain adesired pH and/or adjusting dissolved oxygen concentration to maintain adesired dissolved oxygen concentration and/or adjusting dissolved carbondioxide concentration. The methods can include adjusting dissolvedoxygen concentration and adjusting dissolved carbon dioxideconcentration, whereby in response to pH measurement(s), dissolvedcarbon dioxide levels are adjusted; and/or adjusting dissolved oxygenlevels in response to dissolved oxygen measurement(s). The methods caninclude adjusting pH to a desired level in response to pH measurementsby adjusting the dissolved carbon dioxide concentration such thatdissolved carbon dioxide concentration is adjusted when pH varies fromthe desired level, and the dissolved oxygen measurement variesperiodically as a function of time. The methods can include adjustingthe dissolved oxygen concentration so that the dissolved oxygenmeasurement varies from 30% to 90% or from 40% to 80% or from 50% to70%; or, so that the dissolved oxygen measurement averages about 60%and/or adjusting the dissolved oxygen concentration so that thedissolved oxygen measurement varies from high value to low value overabout 10 to about 30 minutes or over about 20 minutes and/or a plot ofthe dissolved oxygen measurement as a function of time comprises a sinwave. Additionally or alternatively, the adjusting can be an adjustmentof conditions in response to cell density and/or cell count measurement;for instance, media can be added and/or changed and/or a vector (e.g.,recombinant virus such as baculovirus) added for infection in responseto the cell density and/or cell count measurement.

Yet further still, the methods can include collecting the cells. Theinvention thus comprehends methods for producing cells. The inventioneven further comprehends wherein the cells contain a vector. Thus, theinvention also comprehends methods for replication of the vector and/orexpression of exogenous nucleic acid molecules. The vector can comprisea virus or a recombinant virus; e.g., a baculovirus or recombinantbaculovirus. The invention even further comprehends collecting expressedproduct, and/or virus or vector, e.g., baculovirus and/or the cells, aswell as expressed product from the methods.

The invention therefore provides a method, for producing an expressionproduct from a recombinant vector infected or transfected or insertedinto a cell, or for producing a vector infected or transfected orinserted into a cell, comprising performing aforementioned or hereindisclosed methods, wherein cells of the cell culture are infected ortranfected with or have inserted into them the recombinant vector, orthe vector, either prior to or during the method. The recombinant vectorcan be a virus, e.g., a recombinant virus, such as a baculovirus and thecells can be cells susceptible to such a virus e.g., insect cells. Thecells can be infected and/or transfected and/or have the vector insertedtherein during the aforementioned and/or herein disclosed methods, e.g.,during use and/or within inventive apparatus; and, collecting the cellsor the expression product or the recombinant vector or the vector can beincluded.

Accordingly, the invention yet further comprehends uses of theexpression products; e.g., as diagnostics, therapeutics, antigens,epitopes(s) of interest, vaccines, immunological compositions,therapeutic compositions, diagnostic compositions, etc.; and, theinvention comprehends products from such uses, e.g., immunologicaland/or vaccine and/or diagnostic and/or therapeutic compositionscomprising an antigen and/or epitope of interest and/or diagnosticprotein and/or therapeutic wherein the antigen and/or epitope ofinterest and/or diagnostic protein and/or therapeutic is obtained fromherein described methods and/or apparatus, and/or antibodies or antibodycompositions elicited by such an antigen and/or epitope of interest(e.g., from administration of the antigen or epitope to a suitableanimal), as well as methods involving such products, such as methods forinducing an immunological or immune response or protective immuneresponse or therapeutic response comprising administering thecomposition comprising the antigen and/or the epitope of interest and/orthe antibody and/or the therapeutic and methods involving diagnosticproteins from the invention, e.g., contacting a sample with a diagnosticprotein obtained from this invention to ascertain the presence orabsence of an antibody to the diagnostic protien.

The terms “comprises” and “comprising” can have the meaning given theseterms in U.S. Patent Law; e.g., they can mean “includes” or “including”.

Further embodiments of this invention will be set forth in thedescription that follows, and will become apparent to those skilled inthe art and as learned by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, and notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, incorporatedherein by reference, in which:

FIG. 1 shows a schematic illustration of a High-Density DialysisBioreactor with In-Line oxygenation;

FIG. 2 shows a schematic illustration of the cell culturing loop of FIG.1;

FIG. 3 shows a schematic illustration of the medium replenishment loopof FIG. 1;

FIG. 4 shows a schematic illustration of the hollow fiber dialysisdevice of FIG. 1;

FIG. 5 shows a graph describing Growth of Insect Cells in a High-DensityDialysis Bioreactor with In-Line oxygen Sparging;

FIG. 6 shows a bar graph comparing Yields of AcNPV Polyhedrin Protein inStandard and High-Density Cultures;

FIG. 7 shows a bar graph comparing Yields of Recombinant Hemagglutininfrom Three Strains of Viral Influenza in Standard and High-DensityCultures;

FIG. 8 shows a graph comparing the effects of oxygenation on growth;

FIG. 9 provides a Bioreactor diagram legend (legend of components; seeFIGS. 1–4);

FIG. 10 shows a flow diagram with outputs from probes 114 a–e going tomicroprocessor or processor or computer controlling parameters such aspH, carbon dioxide, oxygen, air, nitrogen, temperature and connected tosystem inputs therefor (e.g., 154, 130, 140; heating/cooling e.g., formedia reservoir, for bioreactor) with pH, oxygen, carbon dioxide andtemperature functions illustrated in the flow diagram; and,

FIG. 11 shows CHO cell growth in a high density bioreactor according tothe invention vs. growth in a control flask.

DETAILED DESCRIPTION

A bioreactor/cell culture process desirably provides for at least one ormore, and advantageously all of: rapid growth of cells, preferably tohigh density, nutrient utilization and waste removal, preferablyefficient nutrient and/or waste removal, and optimum accumulation ofbiological substances of interest. “High density” can have the meaninggiven to this term in the art, e.g., literature, patents, such as thosecited herein, and can mean cell densities as exemplified herein, and/orabout ±15% or about ±10% about or ±5% or about ±3% or about ±1% of thesevalues, but higher cell densities, e.g., higher than those reportedherein and/or higher than about 10% or about 15% greater than valuesexemplified herein, are desirable. Advantageously, “normal” density canbe a density achieved without the present invention, e.g., understandard conditions (such as stirred bioreactor with direct sparginginto the bioreactor without circulation of cells or medium), and highdensity can be a 20% or 50% or 100% or 150% or 200% or even a 300%, 400%or 500% or more increase in cells over normal (note the Examples infra).

The apparatus and process of the present invention, while developed forand advantageously employed with respect to lepidopteran insect cells,provides beneficial conditions for many diverse cell types; namely, allcell types, including without limitation, eukaryotic and prokaryoticcells; vertebrate and invertebrate cells; animal and plant cells; fungusor yeast and bacteria cells; for instance, plant cells such as landplant cells and marine plant cells, monocot cells and dicot cells e.g.maize cells, tomato cells, tobacco cells; yeast cells such asSaccharamyces cerevisiae cells, Saccharamyces pastorianus cells Pichiapastoris cells; bacteria cells such as E. coli, Bacillus (e.g.,Lactobacilli), Staphylococci; vertebrate cells such as fish cells (e.g.,shark, salmon, rainbow trout, zebrafish, herring, mackerel cells),amphibian cells (e.g. frog, toad, salamander cells), bird or avian cells(e.g. chicken, turkey, duck, pigeon, dove cells), reptile cells (e.g.snake such as cobra), and mammalian cells (e.g., human, rabbit, hamster,mouse, rat, primate, cells such as VERO, HeLa cells, Chinese hamsterovary (CHO) cell lines, W138, BHK, COS-7, 293, MDCK, blood cells (e.g.,red blood cells and white blood cells)); invertebrate cells such as landinvertebrate cells, for instance, insect cells, e.g., lepidopteran cellssuch as Spodoptera (e.g., Spodoptera frugiperda such as Sf9 or Sf900+ orATCC CRL 12579; see also U.S. Ser. No. 09/169,178, filed Oct. 8, 1998),Trichoplusia (e.g., Trichoplusia ni such as cells as in Granados, U.S.Pat. Nos. 5,300,435, 5,298,418), silkworm (Bombyx mori), dipteran suchas mosquito (e.g. Culicidae) cells, fly cells (e.g. Drosophila),transformed insect cells (see, e.g., Ailor et al., “Modifying secretionand post-translational processing in insect cells,” Current Opinion inBiotechnology 10: 142–145 (1999); Pfeifer et al., “Expression ofheterologous proteins in stable insect cell culture,” Current Opinion inBiotechnology 9: 518–21 (1998); McCarrollet al., “Stable insect cellcultures for recombinant protein production,” Current Opinion inBiotechnology 8: 590–94 (1997); U.S. Pat. No. 5,637,477), and marineinvertebrate cells, for instance shrimp cells (including Penaeus such asPenaeus monodon, P. japonicus and P. penicillatus); e.g., typical cellsthat are used with eukaryotic replicable expression vectors such a S.frugiperda cells, VERO cells, MRC-5 cells, SCV-1 cells COS-1 cells,NIH3T3 cells, mouse L cells, HeLa cells, CHO cells, and the like. Thecells can be recombinant; e.g., the cells can have been infected ortransfected with or by a vector or otherwise have inserted therein avector (e.g., before, during or after use of the cells in the bioreactorsystem and methods of use of the invention), and the vector can containa particular nucleic acid molecule, e.g., a heterologous or exogenousnucleic acid molecule (as to either the cell or the vector or both); forinstance, for reproduction and/or expression of certain nucleic acid(e.g., DNA) molecules.

It is advantageous in growing cells to supply and maintain nutrients andoxygen uniformly or substantially uniformly or with consistency orsubstantially consistently or regularly or substantially regularly, aswell as maintain cell viability, whether in the cell growth or proteinsynthesis phase. Note for instance the regular variation in cell cultureparameters in embodiments of the present invention, or the holding or ofone or more parameters constant or uniform (or substantially constant oruniform).

Embodiments of the present invention demonstrate the applicability ofthe present invention to all cell types because addressing design issuewith respect to insect cells provides teachings to practice theinvention with respect to any cell type, since one can extrapolate frominsect cells to other cells, and insect cells are a true test of theinvention. For instance, insect cells require oxygen over and above whatis required for most animal cells (Maiorella B, Inlow D, Shauger A,Harano D (1988) Bio/Technology 6: 1406). When infected by baculovirus,the oxygen requirement increases yet again (Kiouka N, Nienow A W, EmeryA N, al-Rubeai M (1995) Journal of Biotechnology 38(3): 243). And,improper delivery of oxygen can result in cell damage and ultimately,cell death through shear forces related damage.

The invention provides many advantages. In at least certain embodiments,the invention is simple in that it can include three main components: Acell culture Loop 100. A Medium Replenishment Loop 200. And, HollowFiber Dialysis Device 300. Other embodiments can be simpler.

Further, components of the invention can be modular such that eachmodule can be replaced during the culture process either as a plannedevent such as a requirement for optimal production of a biologicalsubstance, or as an unplanned event such as the failure of a component.This exchange of modules can occur without having to halt the cultureprocess. The simplicity and modularity of the present invention make itflexible in that the invention can accommodate a variety of cultureparameters such as cell type, and scale or process type such as batch orcontinuous.

Further still, the simplicity, modularity and flexibility of theinvention means that it lends itself to automation through the additionof appropriate sensors in the system; for instance as discussed herein,see, e.g., FIG. 10. These could monitor one or more or any combinationor all of: temperature, pH, conductivity, dissolved oxygen, glucoselevel, cell density, carbon dioxide, and nitrogen, for example. Acomputer programmed with the optimum culture conditions can monitor thesensor data and adjust chemical or physical properties, such as pH (forinstance by addition of carbon dioxide) or oxygen (for instance byaddition of oxygen), or temperature, in response to sensor data. Whendeviations from the prescribed conditions are detected, the computerthen automatically would adjust the appropriate culture parameters suchas impeller speed, oxygen flow rate or medium flow rate until theculture conditions once again fall within acceptable ranges. Thus, this“feedback loop” between the sensor data and the computer would allow forunattended operation of the invention.

Advantageous embodiments can include means for dialysis. This means canbe a hollow fiber filter; and, this has been found to be an importantcontribution to improving cell culturing system yields. These slightlyflexible semi-permeable capillary tube devices are usually contained ina rigid encasement. Because they are semi-permeable, that is, they allowsmall molecular size material to pass through their pores whileretaining the much larger intact cells, they are utilized inparticularly advantageous embodiments to separate the desired biologicalproduct from the cells during fermentation. Another means for dialysiscan be a tangential flow filter, i.e., another semi-permeable membraneuseful as a dialysis means in this invention can be a tangential flowfilter.

In certain advantageous embodiments, the dialysis means is present andan interface between the Cell Culturing Loop and the MediumReplenishment Loop. (See FIG. 1: Note that cell culture from bioreactor110 flows through cell take-up and line 112 into line 112 a (throughaction of pump 120), and passes through line 112 b into the Hollow FiberDialysis Device 300 via Lumen input 301. Cell culture from Lumen input301 flows into Lumen space 310 and out Lumen outflow 302 to cell returnline 113 a. Lumen space 310 is within the hollow fiber filter of thehollow fiber filter device 300 (which has a cylindrical shape). Fromline 113 a, cell culture flows into line 113 b, and then passes thoughoptional, but advantageously present, oxygenation loop 150 via lumeninput 152 and lumen outflow 153 (at opposite ends of the lumen 151 a ofoxygenation loop 150), returning to biorecator 110 via line and cellreturn 113. Media from media reservoir 210 flows through media take-up212 into line 250 and to line 250 a (through the action of pump 220) andinto extra lumenal input 303 of the Hollow Fiber Dialysis Device 300.From extra lumenal input 303, media flows into extra lumen space 320,which has an exterior surrounding relationship to the hollow fiberfilter of the Hollow Fiber Dialysis Device 300 and is within the lumenof the Device. The media then flows out extra lumenal outflow 304,through lines 260 c, 260 b, 260 a and 260 back into media reservoir 210via media return 213. Thus, media flows on the outside of the hollowfiber filter while cell culture flows through the interior of the hollowfiber filter, with dialysis occurring as the liquids pass on oppositesides of the filter—nutrients flowing from the media into the cellculture through the hollow fiber filter, waste from the cell cultureflowing into the media through the hollow fiber filter (nutrients andwaste products in the bioreactor and the dialysate are in equilibriumand do not necessitate continuous perfusion (dialysis used not only forremoval of waste but also for addition of nutrients))- and the HollowFiber Dialysis Device is a dialysis means that is an interface betweenthe Cell Culture Loop and the Medium Replenishment Loop.)

Having the dialysis means as an interface between the Cell CulturingLoop and the Medium Replenishment Loop provides advantages. Forinstance, in the practice of embodiments of the present invention, onecan use a hollow fiber filter without: having to remove medium and thecells from the bioreactor vessel, then pass the medium and cells throughthe filtering device, with subsequent collection of the perfused fluidcontaining the desired biological substance and returning the mediumwith its cells to the original bioreactor vessel; or having to housecells of interest within the extra-lumenal space of the device itself,with perfused medium passing through the capillary tubes to the cells;or placing the unencased hollow fibers directly into the fermentationtank itself so that fresh medium can be more directly provided toimmobilized or attached cells.

The inventive bioreactor system and methods of use can in certainadvantageous embodiments involve a combination of improvements thattogether can provide for high-density growth and production ofbiologically important materials. In these embodiments, the design canprovide favorable oxygen, and/or nutrient supplies and reduced shearforces necessary for high-density propagation of cells. Theseembodiments can include: continuous circulation of cells from thebioreactor, through a semi-permeable hollow fiber filter, then back tothe bioreactor; in a manner that is analogous the circulation of theblood through the kidneys and also includes in-line oxygenation, as inthe lungs; medium is pumped from a storage vessel to the hollow fiberfilter and then circulates back to the storage vessel. In the hollowfiber filter, dialysis occurs between circulating replenishment mediaand cells; removing waste products and replenishing nutrients utilizedto support the metabolism of the cells. The method in these embodimentscompartmentalizes the process of culturing cells, and thereby producingimportant biological substances, into three discrete components: onecontaining the cells, one containing a volume of medium and the third asemi-permeable device allowing interaction between the cell compartmentand the medium reservoir compartment. Thus, like circulating bloodcells, cells in this bioreactor system can be maintained underconditions optimal for growth or production of cellular products.

Thus, the invention can involve a bioreactor for containing cellculture, dialysis means, and a media reservoir for containing mediawherein the bioreactor is connected with the dialysis means and themedia reservoir is connected with the dialysis means such that inoperation there is dialysis between the cell culture and the media; and,each of the cell culture and media may be in circulation via circulationor pumping means.

Accordingly, in certain advantageous embodiments, the invention canfurther involve oxygenation means, illustrated in the Figures as anoxygenation loop within the cell culturing loop. The illustratedoxygenation means (see FIG. 1) includes oxygenator 151 that includeslumen 151 a, gas input 154, gas output 155, lumen input 152 and lumenoutflow 153 (with cell culture flowing from line 113 b into lumen input152 at the top of oxygenator 151 and out of the oxygenator at lumenoutflow 153 at the bottom of oxygenator 151 and into bioreactor 110 viacell return 113). The gas input can, of course, be connected to anoxygen source, to provide oxygen to the cell culture; and, other gasescan also be inputted through input 154, e.g., air and/or carbon dioxideand/or nitrogen. Furthermore, an alternative can be that input from line113 b flows into input 154 and output 155 is connected to line 113, withgas introduced at input 152 and exiting at outflow 153; i.e., the portscan be “flipped”. Alternatively or additionally, oxygenation means caninclude introducing (e.g., at the point in FIG. 1 of the oxygenationloop) oxygen and/or an oxygen source or carrier into the cell culture(that diffuses oxygen into the cell culture), such as perfluorocarbonoxygen carriers, hemoglobin, and the like, either alone or incombination with one or more other gases and/or gas sources or carriers.

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Immobil.Biotechnol. 1994; 22(4): 955–963; Spence, R. K. Perfluorocarbons in thetwenty-first century: clinical applications as transfusion alternatives.Artif. Cells Blood Substit. Immobil. Biotechnol. 1995; 23(3): 367–380;Shah, N.; Mehra, A. Modeling of oxygen uptake in perfluorocarbonemulsions: some comparisons with uptake by blood. ASAIO Journal. 1996;42: 181–189; Patel, S., et al. Modeling of oxygen transport inblood-perfluorocarbon emulsion mixtures. Part II: tissue oxygenation.ASAIO Journal. 1998; 44(3): 157–165; Hoffman, R., et al. Arterial bloodgases and brain oxygen availability following infusion of intratrachealfluorocarbon neat liquids. Biomater. Artif. Cells ImmobilizationBiotechnol. 1992; 20(2–4): 1073–1083; Forman, M. B., et al. Role ofperfluorochemical emulsions in the treatment of myocardial reperfusioninjury. Am. Heart. J. 1992 November; 124(5): 1347–1357; Jacobs, H. C.,et al. Perfluorocarbons in the treatment of respiratory distresssyndrome. Semin. 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C., et al. Perfluorocarbons:future clinical possibilities. J. Invest. Surg. 1997 November–December;10(6): 357–365; Goodnaugh L. T., et al. Oxygen carriers as bloodsubstitutes. Past, present, and future. Clin. Orthop. 1998 December;(357): 89–100; Chiba, T., et al. Transabdominal oxygenation usingperfluorocarbons. J. Pediatr. Surg. 1999 May; 34(5): 895–900; discussion900–901.

The dialysis means in embodiments of the inventive bioreactor andmethods of use is by itself believed to be novel. The oxygenator meansin embodiments of the present invention, e.g., oxygen sparging and/orproviding oxygen via an oxygenation loop containing a pore filter, isalso by itself believed to be novel. Thus, embodiments of the inventioncan involve the dialysis means (or dialyzing) without necessarily alsoincluding the oxygenator means. Embodiments of the invention can involveoxygenator means (or oxygenating) without necessarily also includingdialysis means. And, embodiments of the invention can include bothdialysis means and oxygenator means (or dialyzing and oxygenating).(Indeed, dialyzing and oxygenating can be two steps or one step; forinstance, if the media includes not only nutrients but also a source orcarrier of oxygen such that at the dialysis means, nutrients and oxygenboth pass to the cell culture and dialyzing and oxygenating can beperformed in one step.)

Inventive bioreactor systems and methods of use can support the growthof cells, e.g. insect cells, to densities that are higher than thoseknown to the inventors to have ever been reported. Inventive bioreactorsystems and methods of use also produce virus, e.g., baculovirus andrecombinant gene products in cells, e.g., insect cells, at very highcell densities. Furthermore, inventive bioreactor systems and methods ofuse can be employed at large scales and are suitable for the manufactureof recombinant DNA products in cultured cells.

Insect cells from S. frugiperda and other Lepidopteran insect specieshave been described in the literature and their general use to supportthe infection and replication of baculoviruses and recombinantbaculoviruses or insect cell viruses and the production of recombinantproteins therefrom is well known (see, e.g., Smith et al., U.S. Pat. No.4,745,051 (recombinant baculovirus); Richardson, C. D. (Editor), Methodsin Molecular Biology 39, “Baculovirus Expression Protocols” Humana PressInc. (1995)); Smith et al., “Production of Human Beta Interferon inInsect Cells Infected with a Baculovirus Expression Vector,” Mol. Cell.Biol., 3(12): 2156–2165 (1983); Pennock et al., “Strong and RegulatedExpression of Escherichia coli B-Galactosidase in Insect Cells with aBaculovirus vector,” Mol. Cell. Biol., 4(3): 399–406. (1984); EPA 0 370573, U.S. application Serial No. 920, 197, filed Oct. 16, 1986, EPPatent publication No. 265785; U.S. Pat. No. 5,911,982; and otherdocuments cited herein).

In the baculovirus expression system, an inserted nucleic acid molecule,e.g., the foreign gene, the heterologous or exogenous nucleic acidmolecule, for instance, DNA, is inserted into an insect virus vector,e.g., in a baculovirus vector, which is then used to infect cells of theinventive cell line, for expression of the DNA. The DNA preferablyencodes an expression product. Similarly, when the inventive bioreactorprocess is used with the insect cell line infected with a recombinantbaculovirus, at least one polypeptide of interest is produced.

Similarly, other vector systems for the expression of exogenous DNA areknown; for instance, the poxvirus system; see, e.g., U.S. Pat. Nos.4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807,4,722,848, WO 94/16716, WO 96/39491, Paoletti, “Applications of poxvirus vectors to vaccination: An update,” PNAS USA 93: 1349–11353,October 1996, and Moss, “Genetically engineered poxviruses forrecombinant gene expression, vaccination, and safety,” PNAS USA 93:11341–11348, October 1996. In embodiments of the invention, instead ofinsect cells in the inventive bioreactor system and methods of use, onecan use cells susceptible to expressing nucleic acid molecules ofpoxviruses—either heterologous or homologous nucleic acid molecules,e.g., cells susceptible to poxvirus infection and/or cells in which apoxvirus can have expression of at least some gene products (eitherheterologous or homologous gene products) without productive replicationof the virus (e.g., wherein the cell is not naturally a host of theparticular poxvirus such as infecting a mammalian cell with an avianpoxvirus); and, these cells may be infected with a poxvirus or arecombinant poxvirus for reproduction of and/or expression from thepoxvirus (or, one can use insect cells and infect them with an insectpoxvirus or a recombinant insect poxvirus—either one that hasreproduction and/or expression in such insect cells (e.g., wherein theinsect cell is a natural host of the poxvirus) or has expression withoutproductive replication in such insect cells (e.g., wherein the insectcell is not a natural host of the poxvirus)).

Similarly, there are other vector systems such as bacterial, and yeastsystems, minichromoshomes, retrovirus vectors (e.g., murine leukemiaviral vectors), retrotransposons or virus like particles, bovinepapilloma virus vectors, SV40 based vectors, mammalian cell systems,other viral systems e.g. herpes virus systems, adenovirus systems, andDNA plasmid systems, inter alia; see, e.g., U.S. Pat. No. 4,769,331(recombinant herpesvirus), Roizman, “The function of herpes simplexvirus genes: A primer for genetic engineering of novel vectors,” PNASUSA 93: 11307–11312, October 1996, Frolov et al., “Alphavirus-basedexpression vectors: Strategies and applications,” PNAS USA 93:11371–11377, October 1996, Kitson et al., J. Virol. 65, 3068–3075, 1991;U.S. Pat. Nos. 5,591,439, 5,552,143 (recombinant adenovirus), Grunhauset al., 1992, “Adenovirus as cloning vectors,” Seminars in Virology(Vol. 3) p. 237–52, 1993, WO 98/33510, Ballay et al. EMBO Journal, vol.4, p. 3861–65, Graham, Tibtech 8, 85–87, April, 1990, Prevec et al., J.Gen Virol. 70, 429–434, PCT WO91/11525; Ju et al., Diabetologia, 41:736–739, 1998 (lentiviral expression system); Felgner et al. (1994), J.Biol. Chem. 269, 2550–2561, Science, 259: 1745–49, 1993 and McClementset al., “Immunization with DNA vaccines encoding glycoprotein D orglycoprotein B, alone or in combination, induces protective immunity inanimal models of herpes simplex virus-2 disease,” PNAS USA 93:11414–11420, October 1996, and U.S. Pat. Nos. 5,591,639, 5,589,466, and5,580,859 relating to DNA expression vectors, inter alia., Fischbach etal. (Intracel) WO 90/01543 (method for the genetic expression ofheterologous proteins by cells transfection); and Robinson et al.,seminars in IMMUNOLOGY, vol. 9, pp. 271–283 (1997) (DNA vaccines). Cellsuseful with such other vector systems can be employed in the bioreactorsystem and methods of use thereof of the present invention; and, suchcells can be infected or transfected or have plasmids containingexogenous DNA inserted therein, as the case may be depending on the celland vector system, prior to or during or after growth and being employedin the inventive bioreactor and methods of use of the invention, e.g.,for protein production using the inventive bioreactor and methods of usevia those cells and another vector system.

With respect to terms, reference is made to documents cited herein, andgenerally to Kendrew, The Encyclopedia Of Molecular Biology, BlackwellScience Ltd., 1995 and Sambrook, Fritsch and Maniatis, MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1982 (“Maniatis et al., 1982”).

CERTAIN SYSTEMS OF THE INVENTION: Systems and certain advantageousembodiments of the invention can be practiced is illustrated in FIGS. 1to 4, 9 and 10.

As shown in FIG. 1, a system can include three interconnected modules,the cell culturing loop 100, the medium replenishment loop 200 and thehollow fiber dialysis device 300. FIGS. 2–4 show these loops and device,with FIG. 9 listing components in certain advantageous embodiments ofthe invention, and FIG. 10 providing a flow diagram of the processor,microporcessor or computer functions in an embodiment of the invention.

THE CELL CULTURING LOOP: The cell culturing loop 100 can include abioreactor 110 (that contains cell culture or culture in use),advantageously a stirred tank bioreactor, onto which is attached aheadplate assembly 111. This headplate can contain a number of ports112–115 through which the contents of the bioreactor 100 can becirculated, sampled and monitored. Thus, the bioreactor 110 can includeoptional stirring means, illustrated in FIG. 1 by mechanical stirrer 110a that has its motor positioned above bioreactor 110; but, otherstirring means can be employed, such as a magnetic stirrer (as in themedia reservoir; however, the stirrer should not interfere with probesor other devices that may be present and may monitor or controlparameters within the cell culture and a magnetic stirrer may sointerfere as a magnetic field in motion can generate an electrical fieldand such fields could interfere).

In a preferred embodiment, the ports can include a cell take up port 112through which cells in culture are removed from the bioreactor using apump 120, and a cell return port 113 through which the cells arereturned to the bioreactor 110 following circulation, e.g., through thehollow fiber dialysis device 300, the optional at least one or a numberof probe ports 114 to measure and/or control culture conditions (e.g.,probe ports 114 a to 114 e—more or less probe ports can be provided,depending upon how many conditions one wishes to monitor or havecontrolled e.g. monitor and/or control via a processor, computer ormicroproscessor; for instance, there can be a probe for any one, or anycombination, or all of: pH, conductivity, oxygen, carbon dioxide,nitrogen, glucose (and/or other nutrient(s)), ammonia (and/or otherwaste product(s)), temperature, cell density, cell count; and, theseprobe(s) can lead to one or more microprocessor, processor or computer,which in turn can be connected to sources for supplying or altering anyone or all of these parameters, whereby the parameters are altered orsupplied in response to measurements from the probes), the optional atleast one sampling port 115 through which culture aliquots can beremoved e.g., sterilely removed, to microscopically examine the cultureor to directly measure culture metabolites for example, and a vent tube116 that allows for pressure equilization in the bioreactor 110.

The cell culturing loop 100 can also optionally include at least onethree-way valve, illustrated as two three-way valves 130 and 140,through which culture components can either be sterilely added orremoved (e.g., via lines 130 a or 140 a) without having to access thebioreactor directly. Note that addition or removal of culture componentscan occur at either three-way valve. Further, note that these three-wayvalves can be controlled by a processor, microprocessor or computer; forinstance, they can be opened and shut for introduction or removal ofcomponents automatically, e.g., opened automatically for introduction ofcomponents in response to data collected at the sensors/probes 114.

Additionally, illustrated embodiments have the cell culturing loop 100also including the optional oxygenation loop 150 that allows for in-lineaddition of oxygen and/or other gases to the culture. This oxygenationloop contains an oxygenation device 151 that in the preferred embodimentis a hollow fiber oxygenator. In this oxygenator would be a lumen inflowport 152 through which the circulating cell culture would enter thelumen of the oxygenator, a lumen outflow 153 through which thecirculating cell culture would exit the oxygenator, a gas input port 154through which oxygen or a gas mixture containing oxygen would enter theoxygenator, a gas output port 155 through which excess gas would leavethe oxygenator and a selenoid 156 that would control the amount ofoxygen added. Note that the placement of the oxygenation loop is, in apreferred embodiment, such that oxygen is added after culture mediumdialysis in the hollow fiber dialysis device 300 but before thecirculating culture medium is returned to the bioreactor 110. Notefurther that the input at gas input 154 can be automoated, e.g.,controlled by a processor, microprocessor, computer or the like, suchthat gas input 154 can be used for introduction of oxygen and othergases such as nitrogen, air, and carbon dioxide; for instance, inresponse to data from probes/sensors 114. Thus, data from probes/sensors114 can go to a microprocessor, processor or computer, that adjusts gasinput at gas input 154 in response to that data. And, as mentioned,oxygenation means other than the oxygenation device 151 can be employedin the practice of the invention. And, note that as discussed herein,the ports of oxygenation device 151 can be “flipped”; e.g., line 113 bcan flow into input 154 and output 155 can flow to line 113, with gasintroduced at port 152 and exiting at outflow 153.

THE MEDIUM REPLENISHMENT LOOP: The medium replenisment loop can includemedia reservoir 210, a pump (or pumping means or circulating means) 220,an optional valve loop 230 and an optional individual valve 240.

The media reservoir 210 can include closed media vessel 211 (thatcontains media in use) take up line 212 that allows for the media to becirculated from the vessel 211, a vent tube 214 that allows for pressureequalization in the media vessel, and optionally stirring means such asstir bar 215 that agitates the media in the vessel 211. The stir bar 215movement can be powered by a variable speed magnetic motor 216 ontowhich the media vessel 211 is placed; or, there can be other stirringmeans provided, such as a mechanical stirrer that is powered by a motorabove the media reservoir (cf. stirrer 110 a).

The media is circulated from the vessel 211 by pump 220 to the hollowfiber dialysis device 300 (extra-lumenal input 303) via a media outflowlines 250 and 250 a (that are on either side of the pump). After passingthrough the Hollow Fiber Dialysis Device 300, the media exits the Devicevia extra-lumenal outflow 304. From outflow 304, media passes throughlines 260 c, 260 b, 260 a and 260 to media return tube 213, throughwhich media returns to the media vessel 211 (after it has been throughthe hollow fiber dialysis device 300).

The media return path can include optional extraction loop 230 that caninclude one or more and preferably three, three-way valves 231, 232 and233. The first three-way valve 231 can be used to divert the flow ofreturn media to optional line 230 b to the optional second three-wayvalve 232 that can be used to collect (e.g., sample) media after it haspassed through the hollow fiber dialysis device 300 to analyze the mediafor culture metabolites in an in-line fashion. In its default positionthe first three-way valve 231 bypasses the extraction loop 230. Thethird three-way valve 233 serves to direct the media flow back to themain return lines 260 a, 260 and 213 (from valve 232 and line 230 a), orin its default position completes the bypass of the extraction loop 230by the media. Another item in the medium replenishment loop is theoptional sampling three-way valve 240 between lines 260 a and 260(downstream of the extraction loop, between the extraction loop and themedia reservoir) where, for instance, additional media can be obtainedfor analysis (via line 240 a). The default position of this valve 240simply returns the media to the media vessel 211.

Alternatively or additionally, the extraction loop and/or the valve 240can run to or be supplied with (e.g., via line 240 a) a series ofsensors or probes (e.g., glucose, nutrient content, and/or ammonia,waste content, etc.); and, these probes or sensors can be connected to aprocessor or computer or microprocessor that can collect informationand/or be further connected to supply lines for the media or componentsthereof.

For instance, media can come out of line 230 c, be run through yetanother dialysis loop, e.g., to remove waste etc. and increase nutrientconcentration and then return to the medium replenishment loop via valve240. Consider that at predetermined times, valves 231 and 230 can beautomatically opened by a processor, microprocessor or computer, forsampling parameters of the media, e.g., glucose, nutrients, pH,conductivity, etc. and that in response to that data, media can be runthrough line 230 c to a dialysis loop (not shown) for removal of wasteand increase of nutrient concentration and then return to the mediumreplenishment loop via valve 240.

Alternatively or additionally, sensors, probes, etc. at line 230 c cansense glucose/nutrient concentration and/or ammonia/waste concentrationand/or pH and/or conductivity etc., and additional glucose/nutrientsand/or liquid to dilute the media and/or components of the media can beadded via valve 240 and line 240 a, to adjust glucose/nutrientconcentration and/or ammonia/waste concentration and/or pH and/orconductivity etc., in response to the measurements taken at line 230 c;and, this can all be done via a processor, microprocessor or computerconnected to the sensors/probes at line 230 c and the supply line(s) 240a feeding into valve 240. (Indeed, valves 231, 232, 233 and 240, as wellas all valves in the operation of the invention, can be automaticallycontrolled, e.g., controlled by way of a processor, microprocessor,computer, etc.; e.g., at a predetermined time the processor,microprocessor, computer causes valves 231 and 232 to open to allow asample of media to run from valve 231 to line 230 b and then to valve232 and out line 230 c to sensors/probes, for a data sampling, withthose valves subsequently closed for normal operation; and, valve 240would be automatically opened for introduction of any necessarycomponents via line 240 a to adjust the media in response to thereadings from the sensors/probes.)

Thus, a microprocessor, processor or computer could first ask if thetime is such for a sampling of the media, and if yes, then appropriatelyopen valves 231 and 230 for the sampling. The processor can then collectdata regarding pH and/or glucose/nutrient concentration and/orammonia/waste concentration and/or conductivity, etc. and if the datavalues are not in accordance with preset optimum values, then eitherdirect the media through another dialysis loop and send the furtherdialyzed media back to the reservoir via line 240 a and valve 240 or addappropriate components to the media via line 240 a and valve 240.

THE HOLLOW FIBER DIALYSIS DEVICE: The hollow fiber dialysis device iscomposed of a lumen space 310 and an extra-lumenal space 320. In apreferred embodiment, material from the cell culturing loop 100 ispumped through the lumen space 310 and media from the mediareplenishment loop 200 is pumped through the extra-lumen space 320.

CERTAIN PROCESSOR/MICROPROCESSOR/COMPUTER FUNCTIONS: FIG. 10 provides aflow chart of certain functions that can be automated in the practice ofcertain embodiments of the invention.

Data from probe/sensor 114 or 114 a–e, such as any one of or anycombination of or all of pH, oxygen concentration, carbon dioxideconcentration, nitrogen concentration, temperature, conductivity,glucose/nutrient level ammonia/waste level, is fed to processor,microprocessor or computer 1000 that can advantageously be a BioFlo3000or equivalent commercial product; and, the processor, microprocessor orcomputer is connected to sources for ingredients and inputs of thesystem such that the processor, microprocessor or computer can addingredients to the system via inputs in response to data from thesensors/probes.

In step 1001 there is a comparison between the cell culture pH (ccpH)with a set value “A”. “A” can be a pH in the range of about 6 to about7.4, for instance, about 6 to about 7, such as about 6.1 to about 6.7,e.g., about 6.1 to about 6.5, and advantageously about 6.1 to about 6.35such as about 6.25 (an optimal value for certain insect cells employedin exemplified embodiments). “A” can be set to a pH that is optimal forthe particular cells employed in the inventive bioreactor system andmethods of use thereof. In step 1002 the processor, microprocessor orcomputer asks if ccpH does not equal the set value “A” and if so,directs towards adjusting carbon dioxide concentration in the system;that is carbon dioxide is employed to control pH and the trigger is theset value “A”, e.g., about 6.25.

In step 1011 there is a comparison between the cell culture oxygenconcentration (CCO₂) with a set value “B”. “B” can be in the range ofabout 30% to about 90% such as about 40% to about 80%, for instanceabout 50% to about 70%, advantageously about 60% (optimal values forcertain insect cells employed in exemplified embodiments). Thus, “B” canbe greater than 40%, e.g., greater than 40% and can go as high as about90% or even 95%; an advance in the art. “B” can be set to an oxygenconcentration that is optimal for the particular cells employed in theinventive bioreactor system and methods of use thereof (for instance,less oxygen if the cells tend to optimally perform under more anaerobicconditions, and the like). In step 1012 the processor, microprocessor orcomputer asks if ccO₂ does not equal the set value “B” and if so,directs towards adjusting oxygen concentration in the system.

Steps 1002 and 1012 flow to step 1003/1013. Step 1003/1013 directs thesystem as follows: If ccpH>A (e.g., if pH rises above trigger value suchas 6.25), then increase carbon dioxide concentration (e.g., add carbondioxide at input 154); if ccO₂<B, then increase oxygen concentration(e.g., add oxygen at input 154); and, ccO₂ can vary as a function oftime t; e.g., if ccO₂ plotted as a function of time t, with ccO₂=y andt=x, plot can be a sin wave (for instance, the x axis runs through the yaxis at point B, e.g., oxygen concentration of approximately 60%, withthe amplitude being approximately 20% to 30%, e.g., the high point ofthe wave above the x axis can be at about 80% to 90% and the low pointof the wave below the x axis can be at approximately 40% to 30%, withthe oxygen concentration cycling from approximately 80 to 90% toapproximately 40 to 30% over a time of about 10 to about 30 minutes,advantageously about 20 minutes, e.g., there can be two waves—one abovethe x axis and one below the y axis—about every 10 to 30 minutesadvantageously about every 20 minutes, such that if “frequency” in thisinstance is the number of waves that pass a point about 10 to about 30minutes, advantageously about 20 minutes, then the frequency is 2, orthere is a wavelength about 10 to about 30 minutes, advantageously about20 minutes).

Thus, carbon dioxide can be used to control pH, with the trigger beingthe set value for the pH, e.g., about 6.25; and, if the pH rises abovethis value, the carbon dioxide is “turned on”-added to the system. Theaddition of carbon dioxide, of course, reduces the oxygen concentration,and the system allows the oxygen concentration to fluctuate a relativelyconstant amount above and below the set value, or cycle over time (e.g.,about 10–30 min. such as about 20 min), for instance, from about 30 toabout 90% or about 40 to about 80%, with about 60% being a set value(i.e., about 20–30% above 60% and about 20–30% below 60& over a courseof about 10–30 min such as about 20 min). The carbon dioxide thus can beset to 0 to 100%, as it is a variable that is adjusted by themicroprocessor, processor or computer; in contrast to any previousreports advising that carbon dioxide accumulation is a problem. Further,the apparatus and methods of the invention are surprising, especially asinsect cell cultures reportedly do not require HCO₃ ⁻/CO₂ buffering(Karmen et al., supra).

This sin wave or cycling or rhythm or periodicity that has been observedwhen the system is automated can be a function of mechanical or chemicalor biological processes occurring within the system. However, butwithout wishing to necessarily be bound by any one particular theory, itis believed that pH changes can occur due to cellular activites, e.g.,ammonia and lactic acid can be released as wastes from cells, with achange in pH. pH change can trigger the addition of carbon dioxide. Theaddition of carbon dioxide can cause a lowering of the oxygenconcentration. And lowering of the oxygen concentration can cause anaddition of oxygen to the system (or a decrease in the addition of othergases to the system). That is, there can be a cycling of the oxygen viacarbon dioxide adjustments based on pH. The nature of the cycling (e.g.,sin wave vs. another wave such as cosine, amplitude and frequency ofwave, etc.) can be adjusted by varying the set values e.g., for instancethe values for oxygen, and/or pH.

In step 1021 there is a comparison between the temperature of the cellculture, cT, with a set value for temperature, T. At step 1022, thequestion is whether cT<T, and if so, then the microprocessor, processoror computer directs increasing temperature or heat applied and/orreducing cooling. At step 1023, the question is whether cT>T, and if so,then the microprocessor, processor or computer directs decreasingtemperature or heat applied and/or increasing cooling (a heating/coolingjacket can be supplied in-a surrounding relationship to the bioreactorand/or the media reservoir). T can be set to a value that is optimal forthe cells, for instance, depending upon whether the cells function atlow temperatures or high temperatures, such as about 15° to about 55°C., such as about 20° to about 40° or 35° C., advantageously about 25°to about 35° C., for example about 26° to about 30° C. or about 20° toabout 28° C. such as about 24° C. to about 28° C.; and in exemplifiedembodiments about 28° C. (but, like other parameters, e.g., pH, oxygen,etc. temperature is set to a value that is optimal to the particularcell employed in the system).

Thus, as illustrated in FIG. 10, the output from microprocessor,processor or computer 1000 is to the system, e.g., inputs such as 154,130 a, 140 a and heating/cooling for the media reservoir or for thebioreactor. Accordingly, in an embodiment of the invention data fromsensors/probes 114 can be sent to microprocessor, processor or computer1000 that adjusts and/or controls pH, oxygen, temperature and carbondioxide, with set values for these parameters; and, gas input into thesystem is oxygen, carbon dioxide, nitrogen and air. In practice of theinvention, gases from Tech-Air manufactured by BOC Air Co. areadvantageously employed.

Accordingly, in an embodiment of the invention there can besensors/probes and/or controls for oxygen, carbon dioxide, temperatureand pH; or for oxygen, carbon dioxide and pH (e.g., steps 1021, 1022 and1023 can be omitted by the microprocessor, processor or computer; forinstance, system run at room temperature, such as a room maintained at afairly constant temperature).

In further embodiments, nitrogen can be set and adjusted as is optimalfor the cells. Air can be added as is optimal for the cells or inresponse to oxygen and carbon dioxide levels. Further still, glucoseand/or nutrient levels and/or ammonia and/or waste levels and/orconductivity can be measured via sensors/probes 114, with themicroprocessor, processor or computer adding glucose, nutrients, etc. atany or all of inputs 130 a, 140 a and 240 a; that is, themicroprocessor, processor or computer can add to either or both loops ofthe system.

STILL FURTHER EMBODIMENTS: As mentioned, the use of the dialysis deviceis considered novel. Thus, a variation on the present invention can bewherein oxygenation loop 150 of FIG. 1 is omitted (such that line 113 bruns directly into cell return 113). Oxygenation can be omitted in theseembodiments or supplied by alternative means such as by chemical meansadded to the system.

Also as mentioned, use of the oxygenation loop 150 is considered novel.Accordingly, a variation on the present invention can be wherein hollowfiber dialysis device 300 is omitted (such that line 112 b connects toline 260 c and line 250 a connects with line 13 a). In theseembodiments, waste removal can be performed at the end of cell growth orby alternative means.

The invention can be used for producing important biological substancesincluding recombinant proteins, viruses and the cells themselves. Theinvention in advantageous embodiments can provide a cell culture unit, abioreactor; the replenishment medium unit, a reservoir of nutrientmedium; a semi-permeable membrane unit, the hollow fiber filter; and anoxygenation unit, an external source of oxygen and/or other gases.

The invention is advantageously applicable to growing cells such asinsect cells, and generating vectors such as viruses, e.g. baculovirus,for instance, recombinant vectors such as recombinant viruses, e.g.,recombinant baculovirus, and to expression of recombinant proteinstherefrom.

The generation and use of recombinant vectors such as viruses, e.g.,baculovirus, is known; for instance, from documents cited herein,including the patent applications and patents cited herein and documentscited in those patent applications and patents. The conditions limitingthe growth of cells such as insect cells are nutrients, oxygen, and thelevels of growth factors and inhibitors. The nutrient requirements forcells such as insect cells have been studied extensively and a varietyof highly enriched commercial culture media, including serum-free media,have been developed. After nearly 20 years of research into theseimproved media formulations, prior to the present invention, there havebeen no significant improvements have been made on the growth rates,density, or expression levels of cells such as insect cells e.g., S.frugiperda and other lepidopteran insect cells; with only minorimprovements in the yield of vectors such as viruses, e.g.,baculoviruses or vector or virus, e.g., baculovirus gene products.

The S. frugiperda Sf-900+ (also termed herein Sf-900) cell growth isexponential at concentrations as low as 0.5×10⁶ cells/mL up to 6−9×10⁶cell/mL. Interestingly, the cessation of the growth of insect cellsoccurs when the medium is still nutritionally sufficient suggesting thatother factors, such as high levels of cell growth factors or otherfactors, may inhibit cell growth. Even more dramatic is the observationthat infection of Sf-900+ cells with baculoviruses is inhibited at celldensities of 3×10⁶ or higher suggesting again that there are inhibitoryfactors in the media. Also, the oxygen demand increases followinginfection reaching a peak about 2 days post infection of approximatelytwice the oxygen required during cell growth.

Especially advantageous elements of an improved bioreactor system andprocess for the growth of S. frugiperda cells and the production ofrecombinant protein are found in FIGS. 1–4 and 9, and optionally also inFIG. 10: High-Density Dialysis Bioreactor with In-Line oxygen. FIG. 2,shows a stirred cell bioreactor 110 with an outside loop for thecirculation of cells from the bioreactor to a semi-permeable membrane,preferably a hollow fiber filter 300. The cell suspension circulatesthrough the filter, preferably the internal partition (lumen) 310 of ahollow fiber filter, then back to the bioreactor; labeled the CellCirculation Loop in the drawing. Also provided, as shown in FIG. 3, is avessel 210, also with an outside loop for the circulation of mediumthrough a semi-permeable membrane 300, preferably a hollow fiber filter.The medium (called the regeneration medium or media) circulates throughthe filter, preferably the external partition (extra-lumenal) 320 of ahollow fiber filter; called the Media Circulation Loop. The filter isadvantageously semi-permeable, e.g. with pores of up to about 0.60 toabout 0.70, such as about 0.65 μM, in diameter, which excludes cellsfrom passing from the bioreactor to the regeneration medium but allowssmaller molecules like glucose and amino acids or waste products likelactic acid and ammonia to diffuse across the membrane. (The pore sizecan vary depending upon the cells employed in the bioreactor system andprocess of the invention, e.g., smaller pore size for smaller cells.)

The hollow fiber filter (FIG. 3) in this bioreactor system and processacts much like blood vessels in an animal where blood circulatingthrough the gastro-intestinal tract acquires recently absorbed nutrientsand passing through organs like the liver and kidneys where additionalnutrients and metabolic waste products, respectively are added orremoved from circulating blood.

In a preferred embodiment, a second hollow fiber device 150 optimizedfor the exchange of oxygen gas is inserted prior to the cell return portof the bioreactor. It is preferred that oxygen is added to the cellcirculation loop immediately prior to return as this configurationreduces the lag time between the disolved oxygen sensor locatedinternally in the bioreactor and return of oxygenated cells into thebioreactor. This minimizes the possibility of over oxygenating thesystem.

Or, in yet a further alternative embodiment, a second hollow fiberdevice, optimized for the exchange of oxygen gas, is prior to or afterthe hollow fiber filter device employed for medium replenishment.

Or, in another alternative embodiment, in-line oxygen is added directlythrough a valve (e.g., a Y or T valve) in the cell circulation loopimmediately before the hollow fiber filter (such that the hollow fiberfilter is functioning for dialysis between the media and the cellculture and to dissolve oxygen into the system, e.g., oxygen is mixedwith circulating cells and media, as it passes through the lumen of thehollow fibers in the filter device and is carried back to the bioreactoras exceedingly small bubbles or dissolved in the culture medium).

Any excess gas diffuses into the bioreactor tank head space and out avent in the head plate of the bioreactor.

The circulating flow of cells in the cell circulation loop and the flowof regenerating medium in the media circulation loop are advantageouslycontrolled with pumping or circulating means and these can beperistaltic pumps. The two streams can flow either in concurrentdirections or in counter-current directions with equal success. Thepumps can also be controlled by the processor, microprocessor orcomputer, e.g., to adjust flow rate in response to temperature,pressure, or other parameters such as pH, conductivity, amount ofglucose/nutrient or ammonia/waste in system, carbon dioxide, or oxygen.

In certain embodiments described and exemplified herein, the bioreactoris a stirred two liter tank bioreactor (but, the invention is notlimited to this size bioreactor), S. frugiperda insect cells such asSf-900 (also termed herein Sf-900+) are seeded in two liters of cellmedium. The temperature of the cells is maintained at about 20° C. toabout 28° C. such as about 24° C. to about 28 C., e.g., about 27° C. toabout 28° C. and the cells are kept suspended by means of an impellerrotating at about 200 rpm.

During operation, the replenishment medium, housed in a 10 liter glassvessel, is pumped to fluid inlet 303 of the extra-lumenal partition 320of the hollow fiber dialysis device by means of a suitable pump, such asa Masterflex L/S Model 7520-00 with dual Easy-Load II Model 77200-62pump heads with flexible silicone tubing, 6.4 mm i.d. size (Masterflex,size 15).

Medium progresses through the extralumenal chamber, finally exiting thehollow fiber filter device 304 and returning to the medium replenishmentvessel 210 through flexible silicone tubing. Tubing to tubingconnections are Swagelok 8 mm port connectors. Glass to tubingconnections are secured by cable ties. Replenishment medium can beselected from any number of suitable sources including but not limitedto SF-900. An optimum rate of flow through the replenishment medium loopis about 100 ml/min but the process operates satisfactorily at speeds aslow as about 10 ml/min, as high as about 3000 ml/min. Optimum flow ratescan be related to hollow fiber membrane area.

An external vent tube 214 with a filter attached to maintain sterilitycan be regulated as required by means of a clamp or ball valve.

Simultaneously, medium with suspended cells are continuously pumped fromthe stirred tank bioreactor by means of pump such as a Masterflex L/SModel 7520-00 with dual Easy-Load II Model 77200-62 pump heads with 6.4mm i.d. size flexible silicone tubing (Masterflex, size 15).

The optimum rate of flow through the loop is about 100 ml/min but theprocess operates satisfactorily at speeds as low as about 13 ml/minalthough some cell lines begin to settle out in the loop at this speedand at flow rates as high as about 3000 ml/min. above which shear forcesincrease to the point of inducing measurable cell damage. This cellsuspension is first passed through a Y or T valve 130 where viralinnoculum can be added to the cell suspension. The cell suspension nextpasses to the lumen of the hollow fiber filter 310 by way of the lumeninput manifold 301 (A/G Technology, Corp; model CFP-6-D-8A, 0.65 micronpore size, 0.41 m² membrane surface area) where exchange occurs betweenthe nutrient-rich replenishment medium and the cell medium containingmetabolic waste products. The hollow fiber with dialysis,ultrafiltration and microfiltration properties can range in pore sizefrom 30 kD cutoff to 0.65 μM diameter. Filters of pore sizes smallerthan 30 kD cutoff may not provide adequate diffusion while those largerthan 0.65 μM diameter may allow cells to pass through to the mediumreplenishment loop, reducing the activity within the bioreactor(although these parameters can be varied by the skilled artisandepending on the particular cells used or depending on the size of thecells in the bioreactor system and process; e.g., depending uponphysical characteristics of particular cells). The membrane surface areacan range from 0.042 m² to 4.2 m² to provide adequate exchange ofreplenishment nutrients and metabolic waste products in a 1 L culture.

Nutrients pass along a concentration gradient from the replenishmentmedium side of the hollow fiber filter to the cell suspension side ofthe device. Metabolic waste products pass along a concentration gradientfrom the cell suspension side of the hollow fiber filter to thereplenishment medium side of the device. The cell suspension is nextpassed through an oxygenation device 150, such as the OXY-1 hollow fiberoxygenator (UniSyn Technologies). Alternatively or additionally oxygencan also be directly sparged in-line. For instance, oxygenation loop 150can be omitted or supplemented by oxygen directly sparged into thesystem via line 130 a (a selenoid such as selenoid 156 can be added toline 130 a). “In-line sparging” can mean adding oxygen directly into thecirculating cell culture, advantageously upstream or prior to return ofthe circulating cell culture to the bioreactor; and, preferably theoxygen is directly added to the circulating cells prior to or upstreamof any dialysis means. This is in contrast to adding oxygen into thebioreactor. In other embodiments oxygen can be supplemented through themedium recirculation loop or through the hollow fiber filter unit. Inany of these cases oxygen is advantageously maintained at about 60% ofsaturation relative to air (with constant variation permissible asherein discussed). An oxygen probe 114 a can be connected to a controlunit (microprocessor, processor, computer) which can regulate the flowof oxygen through selenoid 156 into input 154. Thus, a simple embodimentcan involve an apparatus as illustrated in FIGS. 1–4 and 9, whereinsensors/probes 114 is includes sensor/probe 114 a connected to a controlunit that regulates the flow of oxygen through input 154 such that theoxygen is advantageously maintained at a substantially constantsaturation or concentration (e.g., sensors/probes/control for pH, carbondioxide can be omitted; sensor/probe/control for temperature may bepresent or omitted, for instance if system run at room temperatureadvantageously in room that is kept at fairly constant temperature).

Depending on the oxygenation site, pressure equalization between thebioreactor and the medium vessel may be required i.e. a line connectingboth vessels' vent ports can be incorporated.

Cells can be returned to the bioreactor in a medium high in oxygencontent and nutrients.

The replenishment nutrient stream returns to the replenishment nutrientvessel with added metabolic waste products and reduced in nutrients.Through the use of valves in the medium recirculation loop, thereplenishment medium vessel can be refilled as needed, either because ofnutrient depletion or waste product accumulation. Or the entire mediumvessel can be replaced with similar of different medium, such asswitching between a growth optimized medium and an expression optimizedmedium.

As mentioned herein, these activities can be automated, e.g., throughthe use of a computer, microprocessor or processor. For instance, asdiscussed, valves 231 and 232 and 233 can be automated, with valves 231and 232 opening at predetermined times for sampling through line 230 c,and based upon the data, additional medum added, and/or the mediumreplaced, and/or the medium further in line filtered or dialyzed. And,as discussed, further in line filtering or dialysis and adding of mediumcan be part of an automated process, e.g., employing valve 240.

The replacement of media too can be automated; for instance, “old” mediacan be removed via valve 232 (e.g., with a flow being from line 260 awith valve 231 open for flow through both lines 260 b and 230 b or onlythrough 230 b or with flow being though line 260 c through valve 231 toline 260 b and valve 233 set for flow to continue to both lines 230 aand 260 a) while “fresh” or “new” or “different” media (as desired)added via valve 240 in a commensurate or sufficient amount relative tothe removal at valve 232, over a period of time. Or, at lines 250 and260 there can be T or Y valves that connect to a second media reservoirand when a particular period of time has passed or particular data issensed e.g., at line 230 c (such as glucose/nutrient and/orammonia/waste concentration), these valves are engaged such that thesystem is in communication with the second media reservoir (either aloneor in conjunction with the first media reservoir). Thus, mediareservoirs (two or more media reservoirs) can be serially connected andactivated for automatic changing of media.

Further, and additionally or alternatively, the replacement of media canbe by means of a “tracer”. More in particular, as “old” media can beremoved via valve 232 (e.g., with a flow being from line 260 a withvalve 231 open for flow through both lines 260 b and 230 b or onlythrough 230 b or with flow being though line 260 c through valve 231 toline 260 b and valve 233 set for flow to continue to both lines 230 aand 260 a) with “fresh” or “new” or “different” media (as desired) addedvia valve 240, the media being added can contain a nutrient, electrolyteor some other chemical or physical moiety that is not deleterious to thesystem, and preferably advantageous to the system (such as a nutrient orelectrolyte beneficial for the cells or a particular cell phase) that isnot present in the “old” media being removed; a tracer.

For instance, the tracer can be a particular nutrient or electrolyte inthe new media that is not in the old media. It can function as a tracerbecause its concentration or how it affects a paramenter, such as pH orconductivity, can be used as a measure for the endpoint of adding newmedia.

Consider, for example, that the tracer is a particular nutrient orelectrolyte that can pass through the dialyzing means into the cellculture. As the concentration of that nutrient in the cell culturereaches a desired value and/or as the pH and/or conductivity of the cellculture changes to a desired value (e.g., as sensed at 114), such isindicative of the “old” media having been sufficiently replaced by the“new” media; e.g., microprocessor, processor or computer obtaining datafrom sensor 114 has a function F1 asking when concentration of tracer(e.g., nutrient and/or electrolyte) “[tracer]” in cell culture mediumand/or cell culture medium pH and/or cell culture conductivity=value “C”(or C1 for tracer and/or C2 for pH and/or C3 for conductivity—e.g.,representative of a desired amount of the nutrient or electrolyte in thecell culture from the new media), then cease adding new media and ceaseremoving old media (stop adding via valve 240 and/or removal at valve232); and, this function F1 can come into play after an earlier functionbegan the process of adding new media and removing old media (thatearlier function can be a function of a period of time having passedfrom the initiation of use of the old media in the media loop, or inresponse to other parameters such as levels of waste and/or nutrient inthe cell culture, e.g., if waste higher than a desired level and/ornutrient lower than a desired level). (F1: C (and/or C1 and/or C2 and/orc3)=[tracer] and/or set pH and/or set conductivity—if yes, then closeremoval and/or addition valves; if no, continue with removal and/oraddition.)

“Tracing” can also be performed exclusively in the media loop. Forinstance, a sensor at the removal valve, e.g., can detect the level ofthe tracer, and the computer, processor, microprocessor cease additionof new media and/or removal of old media based on the level of tracerdetected at that point. In this way, the tracer can be a physical and/orinert entity and/or that which does not pass through or need to pass thedialysis filter.

Moreover, from the foregoing, the invention accordingly comprehends thatthere be at least one media reservoir, e.g., that there can be two or aplurality of media reservoirs. In similar fashion, bioreactors can beserially connected and automated, e.g., for automatically changing orincreasing the cells in the system.

Additionally or alternatively, cell density or cell count can bemeasured at line 140 a or 130 a, and when a certain cell density isachieved, the microprocessor, processor, or computer can allow forintroduction of a vector to infect or transfect the cells (e.g., throughthe other of lines 140 a and 130 a) and/or for changing of media and/oradding of ingredients (new ingredients or additional ingredients) to themedia (via lines as discussed above, e.g., via line 240 a and valve240). For example, the computer, processor or microprocessor can takecell density/count measurement(s) via line 140 a or 130 a at certaintimes; if the measurement equals or exceeds a set value, then the vectoris added (e.g., through the other of lines 140 a and 130 a), so that thecells can be infected and/or transfected, such as with a virus orvector, for instance, a recombinant vector or virus, e.g., abaculovirus. Accordingly, the system can allow for automaticeinfection/transfection at a point of optimal cell density/count. Forexample, at a cell density/count of about 4.5 million or higher, such asat about 5 million or about 10 million or about 15 million or about 16million or about 19 million or about 22 million or higher (e.g., withinsect cells; see, e.g., Examples, infra), the vector can be added. Theskilled artisan, without undue experimentation, can set the optimal celldensity/count level for infection/transfection, from this disclosure andthe knowledge in the art, considering such factors as the type of celland the vector or virus being employed. On this point, it is noted thatWedgewood Technology Incorporated (San Carlos Calif.;www.wedgewoodtech.com/web/index.htm) makes an absorbance probe (modelBT65) that can be used for measuring cell density (as do othercommercial suppliers). The Wedgewood Technology BT65 can be used withtheir model 612 single beam photometer or their model 653 absorbancemonitor. The BT65sensor/653 monitor has analog outputs that can beconnected to a computer, processor or microprocessor via an analog todigital interface (converter), without any undue experimentation. Thus,apparatus for measuring cell density are known in the art (e.g.,absorbance sensors/monitors for measuring cell density) and can be usedin conjunction with the invention (e.g., by connecting outputs from suchunits, for instance via an analog to digital converter or interface to acomputer, processor or microprocessor), without any undueexperimentation. Moreover, the invention comprehends thatinfection/transfection of cells can be automated, as can the replacementor supplementing of media; for instance, on the basis of celldensity/count measurement.

Further, it is noted that the valves 140, 130, 240 and the loop 230 (vialines 140 a, 130 a, 240 a and 230 c, respectively) can be employed forremoving expressed products from the system; e.g., removal of fluid fromone port and replenishment or addition back into system when proteinremoved or with fresh or new fluid added to make up for that removed forproduct removal via another port. For instance, a suitable port can beconnected to a separation means, e.g., a dialysis means or other meansthat may remove the expressed product without disrupting the cells ifthey are present in the fluid and the fluid thereafter returned to thesystem (with or without addition of new or fresh fluid); or a suitableport can be connected to means for processing the cell culture forexpressed product isolation (e.g., means for cell lysis or otherwiseextracting protein from the cell) and means for purifying and/orisolating the expressed product, with replacement added to the systemvia another port.

In addition, apparatus and methods of the invention can be used withother means for increasing cell growth and/or recombinant productexpression, e.g., nutrient media, nutrients, etc. that enhance cellgrowth; promoters such as strong promoters or multiple copies ofinserted exogenous coding nucleic acid (e.g., DNA) that can lead toenhanced expression levels.

A better understanding of the present invention and of its manyadvantages will be had from the following non-limiting Examples, givenas a further description of the invention and as illustration of it.

EXAMPLES Example 1 Growth of Spodoptera Frugiperda (Sf900+) Cells inHigh-Density Dialysis Bioreactor with In-Line Oxygen Sparging

Two liters of S. frugiperda Sf900+ (also called Sf900 in text) insectcells were seeded at 1.5×10⁶ cells/mL (see FIG. 5). Oxygen was suppliedinitially by direct sparging at 60 L/hr and maintained at 60% saturationrelative to air with an oxygen probe in the bioreactor connected to asolenoid regulating the flow of oxygen. The temperature of the cells wasmaintained at 28° C. and the cells were kept in suspension with animpeller rotating at 200 rpm. The pH of the media is generally 6.2. Thecells doubled approximately every 24 hours and were 8.2×10⁶ cells/mL byday 3. On day 3 the cells from the bioreactor were circulated at 100ml/min through silicon tubing connected to the lumen of a hollow fiberfilter (A/G Technology, Corp; model CFP-6-D-8A, 0.65 micron pore size,0.41 ml membrane surface area) then back to the bioreactor with aperistaltic pump (Masterflex L/S Model 7520-00 with dual Easy-Load IIModel 77200-62 pump heads. Using the hollow fiber filter the cellsconcentrated to 1 liter to a density of 16.6×10⁶ cells/ml. An externalvessel with 9 L of media was connected to the second pump head on thesame peristaltic pump and media was circulated through silicon tubing at100 ml/min from the vessel, through the external compartment of thehollow fiber filter, and back to the media vessel. Effective pore sizeof a hollow fiber filter ranges from a lower limit of 0.05 μM to anupper limit of 0.65 μM (30,000 d mol. Wt.) which allows for diffusionacross the membrane without leakage of cells across the filter.Effective flow rates through a hollow fiber filter range from 10 mL/minto 3000 mL/min. Below 10 mL/min cells settle out of suspension and above300 mL/min shear forces begin to disrupt cells. At 4 days the cells wereat 26×10⁶ cell/mL and the oxygen rate was increased to 90 L/hr in orderto maintain the dissolved oxygen at 60% saturation (relative to air). At5.1 days the cell density was 45.9×10⁶ cells/mL and sparging oxygendirectly into the bioreactor was no longer sufficient to keep thedissolved oxygen in the cells at 60%. Direct sparging was stopped andthe oxygen line was connected directly to the circulating cells with aY-connector at a position following the pump and before the hollow fiberfilter. The oxygen flow rate was reduced from 90 L/hr to 9 L/hr. Thisso-called in-line sparging restored the dissolved oxygen level to 60%even with a 10-fold reduction in the oxygen flow rate. The reducedoxygen flow has the added advantages of reducing foaming and associatedcell damage which is minimal in comparison to direct sparging with ahigh rate of oxygen flow.

Sf900+ cells doubled approximately every 24 hours with 97% or higherviability and grew to 74.6×10⁶ cells/ml (FIG. 5). In a similarexperiment where in-line sparging was used throughout the growth ofSf900+ cells in a 3 L bioreactor the cells reached the highest densityevery reported for insect cells of 93.4×10⁶ cells/ml and a viability of97.4%. Cell growth was examined numerically and closely fits anexponential growth curve of the form y=ce where y is the cell density, xis the time, c and b and constants, and e is the natural log. Anexponential curve is show in FIG. 5 that closely fits (R-squaredstatistic equals 0.9189) the growth of the Sf900+ cells in the dialysisbioreactor.

Example 2 Yields of AcNPV Polyhedrin Protein in Standard andHigh-Density Cultures

One liter of Sf900+ cells were infected with AcNPV baculovirus using anMOI of 0.5 pfu/cell at the standard density of 1.5×10⁶ cells/mL or at16.0×10⁶ cell/mL. The high-density culture was maintained in a 3-Literdialysis bioreactor (Applicon) as described in Example 1 with continuousin-line sparging of oxygen at a flow rate of 9 L/hr. The oxygen wasmaintained in the high-density bioreactor throughout infection at theset point of 60% saturation of air. After 4 days the infected cells werecollected and cellular proteins analyzed on SDS-polyacrylamide gels. Thelevels of polyhedrin protein were measured (FIG. 6) using a standardprotein assay (BCA, Pierce). At 1.5×10⁶ cells/ml, 800 milligrams ofpolyhedrin protein were produced per liter of infected cells. In thehigh-density culture over 10,374 mg produced per liter of polyhedrin wasfrom 100 g of wet cells (biomass). This is the highest yield ofpolyhedrin protein ever reported for production in cultured insectcells. The relative yields of polyhedrin protein per gram of cells wasover 100 milligrams/gram, higher than the 62.5 milligrams/gram ofinfected cells produced at the standard density demonstrating that theyield per cell of polyhedrin is actually higher in the high densitycultures compared to standard conditions.

Example 3 Yields of Recombinant Hemagglutinin from Three Strains ofViral Influenza in Standard and High Density Cultures

Sf900+ cells were infected at an MOI of 0.5 with AcNPV baculovirusexpression vectors for A/Texas/36/91, A/Johannesburg/33/94, orA/Nanchang/933/95 viral influenza hemagglutinin at the standard densityof 1.5×10⁶ cells/mL or at 16.0×10⁶ cell/mL in a high-density dialysis3-liter bioreactor as described above in Example 1 and FIG. 1. At 3 dayspost infection the cells were collected and the proteins analyzed onSDS-polyacrylamide gels. Yields of total recombinant hemagglutininproteins were determined using a scanning laser densitometry analysis(LKB Instruments) of the stained gels in comparison to known quantitiesof highly purified A/Texas/36/91, A/Johannesburg/33/94, orA/Nanchang/933/95 recombinant hemagglutinins. The yields of totalrecombinant hemagglutinin from all three strains increased 9.3, 10.1,and 10.1 fold in the high density cultures (FIG. 7) with yields of 840mg/L, 710 mg/L, and 780 mg/L respectively. Although less than the levelsobserved at high cell density for polyhedrin, these yields ofrecombinant glycoprotein per liter are among the highest ever reportedfor any expression system. The yields of rHA per gram of wet cells(biomass) was as high or higher in the high density cultures compared tothe relative yields in standard cultures.

Example 4 Production of Recombinant Baculovirus in High Density Cultures

The inventive high density bioreactor system and process can also beused to produce viruses, for instance, recombinant baculoviruses inSf900+ cells. Table 1 are two examples of the production of infectiousrecombinant baculoviruses in Sf900+ cells infected at a density of about15×10⁶ cells/mL using the inventive bioreactor system and process asdiscussed in Example 1 and FIG. 1.

TABLE 1 Production of Recombinant Baculoviruses Recombinant Cell TiterCell line Baculovirus MOI Density PFU/mL Sf900+ C6274 0.5 15.4 × 10⁶ 2.4× 10⁸ Sf900+ B6989 0.5 15.0 × 10⁶ 8.2 × 10⁸

Example 5 Lack of Cell Aggregation with Sf900+ Cells in High DensityCultures

The degree of aggregation of Sf900+ cells was measured at a low(1.38×10⁶ cells/ml) and in two high-density cultures grown as describedin Example 1 (74.6×10⁶ and 93.4×10⁶ cells/ml). Sf900+ cells were countedusing standard procedures in a hemocytometer. The number of aggregateswith 5 or more cells in a clump and the number of viable and dead cellswere recorded. The cell viability was >98% in both the low andhigh-density cultures. Less than 1.5% of the cells were aggregated inthe low and both of the high density cultures, demonstrating thesurprising result that Sf900+ cells grow in serum-free medium in thehigh-density dialysis bioreactors were essentially as a single-cellsuspension of cells. The fact that Sf900+ cells do not aggregate avoidsthe problem associated with adding reagents or chemicals to the cultureto prevent aggregation. Any aggregation would severely reduce theproductivity of the cells due to diffusional barriers for nutrients orby-products or due to reducing their accessibility to virus infection.

Example 6 Long Term Sustainability of Exponential Growth

One liter of S. frugiperda Sf900+ insect cells were seeded at 3.0×10⁶cells/mL as described in FIG. 8 in a system as described in Example 1and FIG. 1 (“month day” in FIG. 8 means for instance the numerical dayof a month, such that if the month were January, the “month days” inFIG. 8 illustrate readings on the 8^(th), 11^(th), 12^(th), 13^(th),14^(th), 15^(th) and 16^(th) of January—the month—with time zerooccurring on the 8^(th)). Oxygen was supplied initially by directsparging at 6 L/hr and maintained at 60% saturation relative to air withan oxygen probe in the bioreactor connected to a solenoid regulating theflow of oxygen. The temperature of the cells was maintained at 28° C.and the cells were kept in suspension with an impeller rotating at 200rpm. The cells doubled approximately every 24 hours and were 19.4×10⁶cells/mL by day 3. On day 3 the cells from the bioreactor werecirculated at 100 ml/min through silicon tubing connected to the lumenof a hollow fiber filter (A/G Technology, Corp; model CFP-6-D-8A, 0.65micron pore size, 0.41 m² membrane surface area) then back to thebioreactor with a peristaltic pump (Masterflex L/S Model 7520-00 withdual Easy-Load II Model 77200-62 pump heads. An external vessel with 9 Lof replenishment medium was connected to the second pump head on thesame peristaltic pump and media was circulated through silicon tubing at100 ml/min from the vessel, through the external compartment of thehollow fiber filter, and back to the media vessel. At 6 days the cellswere at 35.7×10⁶ cell/mL and the external vessel with 9 L ofreplenishment medium was replaced with a new vessel containing 9 L offresh replenishment medium. At 6.7 days the cell density was 52.2×10⁶cells/mL and sparging oxygen directly into the bioreactor was no longersufficient to keep the dissolved oxygen in the cells at 60%. Directsparging was stopped and the oxygen line was connected directly to thecirculating cell line with a Y-connector at a position subsequent to thepump but ahead of the hollow fiber filter. The oxygen flow rate wasreduced from 6 L/hr to 1.2 L/hr. This so-called in-line spargingmaintained the dissolved oxygen level at 60%.

Sf900+ cells doubled approximately every 24 hours with 97% or higherviability and grew to 91×10⁶ cells/ml (FIG. 8), near to the recorddensity reported in Example 1. Cell growth was examined numerically andclosely fits an exponential growth curve of the form y=ce^(bx) where yis the cell density, x is the time, c and b are constants, and e is thenatural log. A plot of the data and the calculated exponential curve isshow in FIG. 5 that closely fits (R-squared statistic equals 0.9318) thegrowth of the Sf900+ cells in the dialysis bioreactor.

Example 7 Inline Oxygenation

To determine the effect of in line sparging on expression in HD culturestwo cultures were set up containing 22×10⁹ cells which were infectedwith AcNPV baculovirus expression vector for A/Beijing/262/95 viralinfluenza neuramimidase (NA). The culture with standard sparging hadoxygen supplied at 2 L/min through a single 5 mm tube immersed in theculture. The test culture was sparged at 0.2 L/min through the lumenside of the hollow fiber dialysis device. The cultures were harvested 72hours post-infection (hpi) and samples were subjected to SDS-PAGE andwestern blot analysis. Other samples were assayed for NA activity.

A. Culture with Standard Sparging

A 2 L 72 hour old culture of SF+ cells in PSFM medium in a 3 L Applikonfermentor was equipped with the high density apparatus including a 0.16m², 0.651 μm pore hollow fiber filter and a 5 L bottle of PSFM. Cellsand medium were circulated through the filter at 100 mls/min using adouble headed peristaltic pump. Temperature was maintained at 28° C.using a heat blanket, temperature probe and a Valley instrumentscontroller. Dissolved oxygen was maintained at 60% of air using anIngold DO probe and a Valley instruments controller. Oxygen was suppliedthrough a single 5 mm tube positioned directly under the impeller.Agitation was done using a marine impeller spun at 200 rpm.

The cells in this culture grew to a density of 10.8×10⁶ cells/ml(21.6×10⁹ total) in 24 hours. They were infected at an M.O.I. of 0.5with NA innoculum. The culture was harvested 72 hpi at which time itcontained 18.0×10⁹ total cells of which 41% were viable. The culture washarvested by centrifugation at 3000×G for 1 hour. The filter was flushedwith 1 L of the diafiltrate and the cells pelleted at 3000×G for 1 hour.Pellet biomass data is shown below

Volume Pellet HD 1650 ml 77.2 g Diaf wash 1000 ml  3.6 g Total biomass80.8 g

B. Standard Density Control Culture

A 500 ml culture of SF+ cells at 1.5×10⁶ cells/ml was set up in a 3 Lspinner flask and infected at an M.O.I. of 0.5 with NA innoculum. Theculture was collected 72 hpi and the cells pelleted at 3000×G for 1hour. The cell pellet from this culture weighed 4.0 g. Samples from thisculture were subjected to SDS-PAGE and western blot analysis. They werealso tested for NA activity.

C. Culture with in Line Sparging

A 2 L culture of SF+ cells in PSFM medium in an Applikon 3 L fermenterwas configured for a HD culture similar to that for the standardsparging culture. The difference was that this culture was equipped forin line sparging. Instead of oxygen being delivered through a singletube a Y connector w inserted between the cell circulation pump head andthe hollow fiber filter. Oxygen was added through the filter at 0.2L/min while being monitored through the DO probe in the fermentor.

When the cells in this culture grew to a total of 21.9×10⁹ cells (1.9 Lat 11.5×10⁶ cells/ml) they were infected with NA innoculum at an M.O.I.of 0.5. A 100 ml culture of SF+ cells at the standard density of 1.5×10⁵cells/ml in a 250 ml spinner was infected with NA innoculum at an M.O.I.of 0.5 to serve as a control.

Samples for gel analysis were taken at 24, 48 and 72 hpi. The culturewas harvested 72 hpi. The cells were pelleted as above and weighed.

Volume Pellet In line O₂ HD 1900 mls 110.4 g Diaf wash  850 mls  10.2 gTotal biomass 120.6 g

Biomass Summary Table

Note: All biomass values are adjusted for 2 L of culture for comparisonpurposes.

Total Biomass Culture type (g) Standard Culture 20 High Density Culturewith standard sparging 80.8 High Density Culture with in-line sparging120.6

As the biomass data demonstrates, in this example, in-line spargingincreased total cell biomass by approximately 50% even as oxygen wasdelivered at a rate tenfold lower than standard sparging.

Example 8 High Density Growth of CHO Mammalian Cells and Expression ofHuman M-CSF

To measure the ability of the present invention to support the growth tohigh density of cells other than insect cells, such as mammalian cells,and to demonstrate that this can result in improved expression ofrecombinant protein, the following experiment was performed. A Chinesehamster cell line that had been engineered to express human macrophagecolony stimulating factor (M-CSF CHO cells) were obtained from HyCloneLaboratories Inc (Logan, Utah) (cf U.S. Pat. Nos. 5,650,297, 5,567,611,and 4,847,201, inter alia, regarding DNA encoding M-CSF and CHO cellsexpressing M-CSF). The cells were maintained using standard conditionsin shaker flasks on a cell culture shaker (135 rpm) in a 37° C.incubator kept at 5% CO₂ and maintained in HyQ PF CHO medium (HyCloneLaboratories). M-CSF CHO cells were seeded on day 0 at a density of0.9×10⁶ cells/ml in a volume of 1.5 liters in a Bioflo 3000 bioreactor(New Brunswick Scientific, Edison N.J.) and maintained at 37° C. with anagitation speed of 50 rpm, dissolved oxygen set at 50% relative to air,and pH set at 7.3. By day 1 the cells had grown to 1.3×10⁶ cells/ml andthe high-density apparatus of the invention (FIG. 1, Example 1) wasassembled and the cells introduced therein and an experiment accordingto the invention performed. The culture medium from the bioreactor wascirculated at 50 ml/min through the lumen of an 0.45 micron, 0.45 sq ftA&G hollow fiber filter. HyQ PF CHO medium was put into 5 L bottle(dialysis medium) and maintained at 37° C. and circulated at 50 ml/minthrough the hollow fiber filter. On day 3 the 5 L bottle of media wasreplaced with a 5 L bottle of fresh HyQ PF CHO medium and it wasmaintained at 37° C. and circulated at 50 ml/min through the hollowfiber filter. On days 2, 3, 4 and 5 the cells in the high-densitybioreactor doubled about every 24 hours and by day 5 were at a densityof 13.6×10⁶ cells/ml (FIG. 11). In a control flask, 100 ml of M-CSF CHOcells were set up at 0.9×10⁶ cells/ml and maintained under standardconditions for 4 days. The cells doubled approximately every 24 hoursand reached a maximum of 4.6×10⁶ cells/ml on day 3 (FIG. 11).

Therefore, the high-density bioreactor and methods of the inventionproduced at least about 4 times the number of cells per unit volume asunder standard culturing conditions.

M-CSF CHO cells have been engineered to express the human gene producefor M-CSF. Samples of the culture media from day 0 and day 4 in thehigh-density bioreactor and in the control flask were obtained from theexperiment described above and the levels of human M-CSF (Hu M-CSF) weremeasured using a commercial assay kit. The follow Table summarizes thelevels of Hu M-CSF produced by the CHO cells and secreted into theculture media in the high density 2 L bioreactor at days 0 and 4, in the5 L of dialysis media at days 3 and in the 5 L of the second bottle ofdialysis media at day 4, and in the 100 ml control flask at days 0 and4. The total production of Hu M-CSF produced in the control flask was ata level of 3.0 mg/L. Whereas in the high-density bioreactor, a total ofover 11.68 mg/L of Hu M-CSF were produced which represents an increaseof at least over 3.9 times the yield of Hu M-CSF as produced in M-CSFCHO cells maintained under standard conditions. In summary, a mammalianCHO cell line grew to at least about 4 times the cell density andproduced at least about 3.9 times the levels of secreted Hu M-CSF.

Thus, the invention is applicable to various cell lines and can resultin increased cell density and/or increased protein expression.

Cells/ Hu M-CSF Volume ml × mg/L Cell Culture Source (ml) Days 10E6culture High density bioreactor 2000 0 0.9 0.51 bioreactor (2L) dialysismedia 5000 3 7.8 5.12 bioreactor 2000 4 13.6 >4.0* dialysis media 5000 413.6 2.56 Control flask TOTAL >11.68 (100 ml) Hu M-CSF flask 0 1.3 0.38flask 4 4.1 3.0 TOTAL 3.0 Hu M-CSF *Expression was so high that it wasgreater than 4 and off the scale of the assay; it is expected that theincrease is at least 4-fold and can be 10-fold or more.(*Expression was so high that it was greater than 4 and off the scale ofthe assay; it is expected that the increase is at least 4-fold and canbe 10-fold or more.)

Example 9 Human Erythropoietin

The sequence of human erythropoietin (EPO) is available from GenBank(accession no. X02157). The human EPO gene isolated from a genomiclibrary in bacteriophage Lambda EMBL-3 was used as template to amplifyEPO coding sequences by PCR. A construct was made in which EPO's naturalsignal peptide was replaced by a baculovirus signal peptide. A 5′ PCRprimer was made that began at the N-terminal residue of the maturepeptide. A 3′ primer was designed to terminate after the natural stopcodon of the EPO open reading frame. After PCR amplification, theresulting EPO gene fragment was inserted into the pMGS12 baculovirustransfer plasmid using standard procedures (Sambrook, J, Fritsch, E. F.,and Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2nd ed.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Theresulting transfer plasmid contained the coding region from EPOdownstream of the polyhedrin promoter, flanked by AcNPV DNA from theEcoRI “I” fragment (Summers and Smith. A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, May 1987, TexasA&M University). Confirmation of the correct EPO coding sequence (Jacobset al. Nature 313 806–10 (1985)) was determined by DNA sequenceanalysis.

Genomic baculovirus DNA and the transfer plasmids containing the EPOgene were mixed, co-precipitated with calcium chloride, and Sf900+ cells(ATCC CRL-12579, deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2009, under theterms of the Budapest Treaty, under ATCC Designation on Sep. 18, 1998)were transfected as described (Summers and Smith 1987, supra).Recombinant viruses were identified by plaque morphology and severalwere further plaque purified. Recombinant viruses capable of expressingEPO in infected Sf900+ cells were identified and used as baculovirusexpression vectors to produce recombinant EPO in Sf900+ cells.

Sf900+ cells, at a cell density of 1.5×10⁶ cells/ml are infected withthe baculovirus expression vector containing the EPO gene at an MOI of1.0. Sf900+ cells are harvested by centrifugation 72 hours postinfection. The cell pellet is discarded and the supernatant containingsecreted recombinant EPO (“rEPO”) is stored at 4.degree. C. for furtherprocessing.

Product purification follows centrifugation, filtration andchromatographic procedures analogous to those presented for influenzavirus hemagglutinin (U.S. Pat. No. 5,762,939 and allowed U.S.application Ser. No. 08/453,848, incorporated herein by reference).Thus, EPO can be obtained which is purified to substantial homogeneityor to at least 95% purity. With respect to EPO, DNA encoding EPO andsubstantial homogeneity of EPO, reference is also made to Lin, U.S. Pat.Nos. 4,703,008, 5,441,868, 5,574,933, 5,618,698, 5,621,080, and5,756,349. In addition, reference is also made to Wojchowski et al.,“Active Human Erythropoietin Expressed in Insect Cells, Using aBaculovirus Vector: A Role For N-Linked Oligosaccharide”, Biochimica etBiophysica Acta 910: 224–32 (1987), Quelle et al., “High-LevelExpression and Purification of a Recombinant Human ErythropoietinProduced Using a Baculovirus Vector”, Blood, 74(2): 652–57 (1989),Quelle et al., “Phosphorylatable and Epitope-Tagged HumanErythropoietins: Utility and Purification of Native Baculovirus-DerivedForms”, Protein Expression and Purification 3: 461–69 (1992), and U.S.Pat. Nos. 5,322,837 and 4,677,195. In contrast to any prior EPO frombaculovirus expression, EPO in accordance with the present invention canbe purified to at least 95% purity or to substantial homogeneity; and,the EPO in accordance with the present invention is produced inrelatively high amounts, is glycosylated and secreted, and has physicaland biological properties as follows: 25 kD, secreted monomers;stimulates erythropoiesis, stimulates erythropoiesis.

As a particular purification procedure, centrifuged culture supernatantcontaining rEPO is pH adjusted to pH 8.0 with Tris-base. Proteinatiousand non-proteinatious materials bind to precipitating salts, mainlycalcium hydroxide, and are removed by centrifugation while rEPO remainsin the supernatant. The resulting rEPO containing supernatant isdiafiltered into 10 mM Tris-Cl buffer pH 8.0.

The diafiltered rEPO containing supernatant is applied onto DEAESepharose and equilibrated with 10 mM Tris-Cl buffer pH 8.0. The rEPObinds weakly and is recovered in the flow-through while contaminantsremain bound to the column. Diafiltration into low-conductivity bufferprior to anion-exchange chromatography ensures stronger binding ofcontaminants and higher degree of purification at this step. Thecollected DEAE flow-through is diafiltered into 10 mM sodium malonatebuffer pH 6.0 and applied to CM Sepharose equilibrated with the 10 mMsodium malonate pH 6.0 buffer. The rEPO binds to CM Sepharose whilecontaminants flow through the column. The column is then washed with 10mM sodium malonate buffer pH 6.0 containing 100 mM NaCl, to furtherremove contaminants. The elute rEPO from the column, a 10 mM sodiummalonate buffer pH 6.0 containing 150 mM NaCl is used.

The eluant containing rEPO is applied to a second CM Sepharose columnequilibrated with 10 mM sodium malonate buffer pH 6.0. It is then washedwith 10 mM sodium phosphate buffer pH 7.0 and finally, rEPO is eluted inPBS (10 mM sodium phosphate, 150 mM NaCl).

The EPO expressed is glycosylated and has a molecular weight ofapproximately 25 kD. The amino acid sequence is the same as or analogousto that set forth in literature and patents cited herein. It is quitesurprising that the EPO in accordance with the present inventionstimulates erythropoiesis as the inventive EPO has glycosylation whichdoes not include sialic acid residues, and there is no O-glycosylationbecause the EPO is from baculovirus expression; and, any reportedrecombinant EPO from baculovirus expression heretofore was reported ashaving no such activity.

In particular, urinary EPO (also known as uEPO) and recombinant EPOproduced in mammalian cells are heterogenously glycosylated with complexN- and O-linked oligosaccharides, including sialic acid N-terminalresidues, and are acidic proteins, whereas EPO from recombinantbaculovirus expression can have a comparably simple saccharideconstitution and relative homogeneity, with no sialic acid residues,neutral high-mannose moieties predominating and the highly basic chargedensity of EPO retained, because of the limited capacity of insect cellsto process N-linked oligosaccharides.

Certain literature such as Quelle et al., Blood, supra, at 656,indicates that EPO from expression by insect cells infected withrecombinant baculovirus containing DNA coding for EPO is notbiologically active due to the lack of sialic acid residues. Further,there is a body of literature asserting that EPO's “heavy glycosylation”and sialic acid residues are essential for biological activity, see,e.g., Marmont, Tumori 83(4 Suppl 2): S3–15 (1997), Morimoto et al.,Glycoconj J 13(6): 1013–20 (1996), Higuchi et al., J. Biol. Chem.267(11): 7704–9 (Apr. 15, 1992), Takeuchi et al., Glycobiology1(4):337–46 (1991), Tsuda et al., Eur. J. Biochem. 188(2): 405–11(1990), Takeuchi et al. J. Biol. Chem. 265(21): 12127–30 (1990), Fakudaet al., Blood 73(1): 84–9 (1989); Matsumoto et al. Plant Mol. Bio.27(6): 1163–72 (1995) (EPO from 9 tobacco cells lacking sialic acidresidues lacked activity).

In contrast, the recombinant EPO of the present invention has anactivity of at least 200,000 U/mg (indeed about 500,000 U/mg) andstimulates erythropoiesis. In further contrast to prior EPO, the EPO ofthe present invention can be isolated using anion exchange and cationexchange chromatography, as opposed to reverse chromatography (used forisolating prior EPO).

Thus, the recombinant EPO of the present invention is distinct from andsurprisingly superior to prior EPO.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1. A method for producing a substantially pure, recombinant,glycosylated erythropoietin (EPO) that has in vivo activity, includingstimulating erythropoiesis, comprising: a) infecting Spodopterafrugiperda insect cells that grow in serum-free media with a baculovirusexpression system comprising a recombinant baculovirus that comprisesDNA coding for EPO such that the recombinant EPO is expressed; b)culturing the infected insect Spodoptera frugiperda cells in serum-freemedia; and c) purifying the recombinant EPO 10 95% or greater purity,whereby the substantially pure, recombinant, glycosylated EPO that hasin vivo activity of between 200,000 U/mg protein and 500,000 U/ mgprotein, including stimulating erythropoiesis, is produced.
 2. A methodfor producing substantially pure, recombinant, glycosylatederyrhropoietin (EPO) that has in vivo activity, including stimulatingerythropoiesis, comprising: a) infecting Spodoptera frugiperda SF900+insect cells that grow in serum-free media with a baculovirus expressionsystem comprising a recombinant baculovirus that comprises DNA codingfor EPO such that the recombinant EPO is expressed; b) culturing theinfected Spodoptera frugiperda SF900+ insect cells in serum-free media;and c) purifying the recombinant EPO to 95% or greater purity, wherebythe substantially pure recombinant, glycosylated EPO that has in vivoactivity, including stimulating erythropoiesis, is produced.
 3. A methodfor producing substantially pure, recombinant, glycosylatederyrhropoietin (EPO) that has in vivo activity, including stimulatingerythropoiesis, comprising: a) infecting Spodoptera frugiperda insectcells that grow in serum-free media with a baculovirus expression systemcomprising a recombinant baculovirus that comprises DNA coding for EPOsuch that the recombinant EPO is expressed; b) culturing the infectedSpodoptera frugiperda insect cells in serum-free media; and c) purifyingthe recombinant EPO to 95% or greater purity, whereby the substantiallypure recombinant, glycosylated EPO that has in vivo activity, includingstimulating erythropoiesis, is produced, wherein the infecting of theinsect cells with the recombinant baculovirus, the culturing of theinsect cells, or both is in an apparatus for growing cells, wherein theapparatus comprises: (i) at least one bioreactor for cell culture; (ii)at least one vessel for culture media; whereby the bioreactor and vesselare in fluid communication, and wherein the bioreactor, vessel, or bothare optionally stirred; (iii) a dialysis means for circulating culturemedia, cell culture, or both, whereby there is a first cell culture loopbetween the bioreactor and the dialysis means and a second mediareplenishment loop between the vessel and the dialysis means; (iv)in-operation dialysis between the culture media and the cell culture;(v) at least one means for delivery of oxygen comprising a hollow fiberfilter oxygenator, whereby the oxygen is delivered directly to cells ina circulating loop of cells before cell entry into the hollow fiberfilter.
 4. The method of claim 3, wherein in the apparatus the means fordelivery of oxygen comprises at least one or more of the following: a)means for in-line sparging; b) means for delivery of at least oneoxygen-containing compound that releases dissolved oxygen into cellculture; c) means for delivery of oxygen positioned upstream of input ofcirculating cell culture returning to the bioreactor; d) means fordelivery of oxygen providing an average dissolved oxygen concentrationof about 60%; e) means for delivery of oxygen providing an averagedissolved oxygen concentration of greater than about 40%; and, f) meansfor delivery of oxygen providing an average dissolved oxygenconcentration between about 30% and about 90%, between about 40% andabout 80%, or between about 50% and about 70%.
 5. The method of claim 3,wherein in the apparatus the means for delivery of oxygen comprises atleast one or more of the following: a) means for in-line sparging; b)means for delivery of at least one oxygen-containing compound thatreleases dissolved oxygen into cell culture; c) means for delivery ofoxygen positioned upstream of input of circulating cell culturereturning to the bioreactor.
 6. The method of claim 4, wherein in theapparatus the dialysis means comprises at least one semi-permeablemembrane, at least one means for delivery of oxygen into the cellculture loop, or both.
 7. The method of claim 4, wherein the apparatusfurther comprises one or more of the following: a) means for measuringphysical parameters of the cell culture or the cell culture media; b)means for measuring chemical parameters of the cell culture or theculture media; c) means for measuring dissolved oxygen concentration; d)means for measuring pH; e) means for measuring pH and dissolved oxygenconcentration; f) means for measuring temperature; g) means formeasuring cell density or amount of cells; h) means for adjustingphysical parameters of the cell culture or the cell culture media inresponse to data from the measuring means; i) means for adjustingchemical parameters of the cdl culture or the culture media in responseto data from the measuring means; j) means for adjusting dissolvedoxygen concentration; k) means for adjusting pH; l) means for adjustingtemperature; m) means for adjusting dissolved carbon dioxideconcentration; and n) means for adding a vector in response to a celldensity or cell amount measurement.
 8. The method of claim 4, wherein inthe apparatus pH is measured, and in response to the pH measurement,dissolved carbon dioxide concentration is adjusted.
 9. The method ofclaim 4, wherein in the apparatus dissolved oxygen concentration ismeasured, and in response to the dissolved oxygen measurement, thedissolved oxygen concentration is adjusted.
 10. The method of claim 4,wherein in the apparatus dissolved oxygen concentration is measured, pHis set to a desired level, and carbon dioxide is adjusted when pH variesfrom the desired level, whereby the dissolved oxygen concentrationvaries periodically as a function of time.
 11. The method of claim 4,wherein in the apparatus the dissolved oxygen concentration is measured,and the measurement varies from 30% to 90%, from 40% to 80%, from 50% to70%, or averages about 60%.
 12. The method of claim 4, wherein in theapparatus the dissolved oxygen concentration is measured, and themeasurement varies from high value to low value over about 10 to about30 minutes, or over about 20 minutes.
 13. The method of claim 4, whereinin the apparatus the dissolved oxygen concentration is measured, and aplot of the dissolved oxygen concentration measurement as a function oftime comprises a sin wave.
 14. Substantially pure, recombinant,glycosylated EPO produced by a method as recited in anyone of claim 1,2, 3 or 13.